He was born in Arundel, Quebec, the son of the Reverend Dr. Dawson Matheson and his wife Gertrude. H17417 Matheson underwent training at the Royal Military College of Canada in 1936.Matheson graduated from Queen's University in 1940, winning the prestigious Tricolour Award in that year for distinguished achievement.

Matheson served as an officer with the 1st Field Regiment, Royal Canadian Horse Artillery, 1st Canadian Infantry Division in Italy during the Second World War where he was wounded. He held honorary militia appointments with the 30th Field Artillery Regiment, Royal Canadian Artillery from 1972 to 1982. He later retired with the rank of Colonel.

After the war, Matheson met Edith Bickley, a radiologist's assistant, in St. Anne de Bellevue Hospital in Montreal, Quebec. He said they would never have met if she hadn’t been such a curious nurse. The couple married and eventually had six children. He received a Bachelor of Laws degree from Osgoode Hall Law School, a Master of Arts degree from Mount Allison University, and a Master of Laws degree from the University of Western Ontario. He was called to the Bar of Ontario in 1948 and was created a Queen's Counsel in 1967. He practiced law with the firm of Matheson, Henderson & Hart in Brockville, Ontario. John now resides in Kingston, Ontario in the St. Lawrence Place retirement residence.

He was first elected as a Liberal in the Ontario riding of Leeds in a by-election in 1961. He was re-elected in 1962, 1963, and 1965. He was defeated by 4 votes in 1968. From 1966 to 1968, he was the Parliamentary Secretary to the Prime Minister, Lester Pearson.

Matheson was a leading member of the multi-party parliamentary committee whose mandate was to select a new flag design. It was George F.G. Stanley's idea that the new flag should be red and white, and that it should feature the single maple leaf. Matheson wrote a book, Canada's Flag: A Search for a Country, about the creation of the new flag.

Matheson also played an important role in the creation of the Order of Canada.

In 1968, he was appointed a judge of the Judicial District of Ottawa-Carleton. In 1984, he was appointed a judge of the County Court of Lanark. In 1985, he was appointed a judge of the District Court of Ontario. From 1990 to 1992, he was a justice of the Ontario Court of Justice (General Division).

A member of the United Church of Canada, Matheson currently lives in Kingston, Ontario.

Matheson was portrayed by Peter MacNeill in a Heritage Minute television commercial about his involvement in the Flag committee.

The John Matheson Sword is awarded annually to the Preparatory Year cadet at the Royal Military College Saint-Jean who achieved the highest results in all four components of the College’s program, namely Academics, Leadership, Athletics and Bilingualism.