George Washington Parke Custis, grandson of Martha Washington, acquired the land that now is Arlington National Cemetery in 1802, and began construction of Arlington House. The estate passed to Custis' daughter, Mary Anna, who had married United States Army officer Robert E. Lee. Custis' will gave a "life inheritance" to Mary Lee, allowing her to live at and run Arlington Estate for the rest of her life but not enabling her to sell any portion of it.[2] Upon her death, the Arlington estate passed to her eldest son, George Washington Custis Lee.[2]

When Virginia seceded from the Union at the start of the American Civil War, Robert E. Lee resigned his commission on April 20, 1861, and took command of the armed forces of the Commonwealth of Virginia, later becoming commander of the Army of Northern Virginia.[3] On May 7, troops of the Virginia militia occupied Arlington and Arlington House.[4] With Confederate forces occupying Arlington's high ground, the capital of the Union was left in an untenable military position.[5] Although unwilling to leave Arlington House, Mary Lee believed her estate would soon be invested with federal soldiers. So she buried many of her family treasures on the grounds and left for her sister's estate at Ravensworth in Fairfax County, Virginia, on May 14.[6][7] On May 3, General Winfield Scott ordered Brigadier General Irvin McDowell to clear Arlington and the city of Alexandria, Virginia, of all troops not loyal to the United States.[8] McDowell occupied Arlington without opposition on May 24.

At the outbreak of the Civil War, most military personnel who died in battle near Washington, D.C., were buried at the United States Soldiers' Cemetery in Washington, D.C., or Alexandria Cemetery in Alexandria, Virginia, but by late 1863 both were nearly full.[10] On July 16, 1862, Congress passed legislation authorizing the U.S. federal government to purchase land for national cemeteries for military dead, and put the U.S. Army Quartermaster General in charge of this program.[10] In May 1864, Union forces suffered large numbers of dead in the Battle of the Wilderness. Quartermaster General Montgomery C. Meigsordered that an examination of eligible sites be made for the establishment for a large new national military cemetery. Within weeks, his staff reported that Arlington Estate was the most suitable property in the area.[10] The property was high and free from floods (which might unearth graves), it had a view of the District of Columbia, and it was aesthetically pleasing. It was also the home of the leader of the armed forces of the Confederate States of America, and denying Robert E. Lee use of his home after the war was a valuable political consideration.[11] The first military burial at Arlington (a white soldier, William Henry Christman) was made on May 13, 1864.[12] close to what is now the northeast gate in Section 27.[13] However, Meigs did not formally authorize establishment of burials until June 15, 1864.[14] The date or name of the first African American burial cannot be precisely determined, but occurred on either July 2 or July 3, 1864, in Section 27.[15] Arlington did not desegregate its burial practices until President Harry S. Truman issued Executive Order 9981 on July 26, 1948.

The government acquired Arlington at a tax sale in 1864 for $26,800, equal to $390,000 today.[17] Mrs. Lee had not appeared in person but rather had sent an agent, attempting to pay the $92.07 in property taxes (equal to $1,400 today) assessed on the estate in a timely manner.[18] The government turned away her agent, refusing to accept the tendered payment. In 1874, Custis Lee, heir under his grandfather's will passing the estate in trust to his mother, sued the United States claiming ownership of Arlington. After the U.S. Supreme Court ruled 5-4 in Lee's favor in United States v. Lee, deciding that Arlington had been confiscated without due process,[18] Congress returned the estate to him. The next year, Custis Lee sold it back to the government for $150,000 (equal to $3,135,909 in 2013) at a signing ceremony with Secretary of War Robert Todd Lincoln.[17]

The southern portion of the land now occupied by the cemetery was used during and after the Civil War as a settlement for freed slaves. More than 1,100 freed slaves were given land at Freedman's Village by the government, where they farmed and lived during and after the Civil War. They were evicted in 1888 when the estate was repurchased by the government and dedicated as a military installation.

President Herbert Hoover conducted the first national Memorial Day ceremony in Arlington National Cemetery, on May 30, 1929.

Beginning in 1992, Morill Worcester donated thousands of wreaths around the end-of-year holiday season to be placed on graves at Arlington. He has since expanded his effort, now known as Wreaths Across America, and supplies wreaths to over 230 state and national cemeteries and veterans monuments across the country.

The first soldier to be buried in Arlington was Private William Henry Christman of Pennsylvania on May 13, 1864.[78] As of May 2006, there were 367 Medal of Honor recipients buried in Arlington National Cemetery,[citation needed] nine of whom are Canadian.

Five state funerals have been held at Arlington: those of Presidents William Howard Taft and John F. Kennedy, his two brothers, Senator Robert F. Kennedy and Senator Edward "Ted" Kennedy, and General of the Armies John J. Pershing. Whether or not they were wartime service members, U.S. presidents are eligible to be buried at Arlington, since they oversaw the armed forces as commanders-in-chief.

Among the most frequently visited sites in the cemetery is the grave of President John F. Kennedy, who is buried with his wife, Jacqueline, and two of their children. His remains were interred there on March 14, 1967, a reinterment from his original Arlington burial site, some 20 feet (6.1 m) away, where he was buried in November 1963. The grave is marked with the "eternal flame". The remains of his brothers, Senator Robert F. Kennedy and Senator Edward M. "Ted" Kennedy, are buried nearby. The latter two graves are marked with simple crosses and footstones. On December 1, 1971, Robert Kennedy's body was reinterred 100 feet (30 m) from its original June 1968 burial site.