The effect of cultivar, curing, storage, tissue type, and cooking method on the ascorbic acid (AA), thiamin, riboflavin, and vitamin B6 content of sweetpotato was determined. A simplified and sensitive reverse-phase high performance liquid chromatography (HPLC) methodology was developed for the simultaneous determination of thiamin and riboflavin in sweetpotato. Curing of sweetpotatoes did not significantly change the content of AA, thiamin, and vitamin B6, but resulted in a decrease in riboflavin content. Thiamin and riboflavin contents were mostly stable after curing. However, compared to at harvest, storage for 6 months resulted in a decrease in AA content in cultivars 07-146, Covington, and Beauregard; and a gradual increase in vitamin B6 content in 07-146, Orleans, and Covington. Although 07-146 contained higher vitamin B6 content, no cultivar was superior or inferior for all the vitamins throughout 6 months of storage. Exposure of sweetpotatoes to chilling injury temperatures of 1 °C and 6 °C for 2 or 4 weeks did not result in consistent changes in AA, thiamin, and riboflavin. However, transfer of the low temperature-stored roots to 14 °C for an additional 7 days generally resulted in AA decreases and stable thiamin and riboflavin contents. Water-soluble vitamin concentration differed between tissue types. Leaf tissue contained no detectable amounts of thiamin, but contained the highest concentrations of AA, riboflavin, and vitamin B6. Cooking methods, including microwaving, boiling, and baking resulted in lower AA compared with raw tissue, but in little differences in thiamin, riboflavin, and vitamin B6. The overall results of this research suggest that while AA is detrimentally affected during commonly used sweetpotato cooking methods, and during typical storage conditions, thiamin, riboflavin, and vitamin B6 contents remain mostly stable. Additionally, they confirm previous reports indicating vegetative tissues can be a good source of AA and multiple B vitamins in human diets.