Born in Monroe in Halifax County, Virginia, he moved with his parents to Mount Zion in Carroll County, Georgia. He attended the common schools and Princeton College and studied law, gaining admission to the bar in 1820 and commencing practice in Sparta, Georgia. Late in 1820, he was chosen brigadier general of the state militia, despite being only 21 years old. In 1823, he married Nancy H. Lane, the daughter of a future presidental candidate, Joseph Lane. Colquitt moved to the village of Cowpens in Walton County and was elected judge of the Chattahoochee circuit in 1826, being reelected three years later. He was licensed as a Methodist preacher in 1827, becoming extremely popular in Central and South Georgia, mostly for his strong support of states rights. He was a member of the Georgia Senate in 1834 and 1837.

Colquitt was elected as a Whig to the Twenty-sixth Congress and served from March 4, 1839, to July 21, 1840, when he resigned. He was elected as a Van Buren Democrat to the Twenty-seventh Congress to fill in part vacancies caused by the resignations of Julius C. Alford, William C. Dawson, and Eugenius A. Nisbet. Following the death of his first wife, Colquitt married Mrs. Alphea B. (Todd) Fauntleroy in 1841, then when tragedy struck again, he married Harriet W. Ross the following year. He was then elected as a Democrat to the U.S. Senate and served from March 4, 1843, until his resignation in February 1848. While in the Twenty-ninth Congress, Colquitt was chairman of the Committee on the District of Columbia and the Committee on Patents and Patent Office. He supported the Polk administration in the controversy relative to the Oregon Territory, and was a prominent opponent of the Wilmot Proviso throughout the Mexican War.

Colquitt retired from national politics in 1848 and resumed his law practice and preaching. He was a member of the Nashville Convention in 1850, arguing for secession if slavery was restricted in any of the new territories then being added to the country. Colquitt died on a trip from Columbus to Macon, Georgia, in 1855. He was buried in Linwood Cemetery in Columbus, Georgia.

Colquitt County, Georgia is named in memory of Walter T. Colquitt. His son, Alfred Holt Colquitt, was also a U.S. Representative and Senator from Georgia, as well as a general in the Confederate States Army during the American Civil War.