Specific separation of thiamine hydrochloride from riboflavin, nicotinic acid, calcium D-pantothenate, pyridoxine hydrochloride, cyanocobalamin, and ascorbic acid has been achieved on commercial precoated silica gel 60F 254 TLC plates with dioxane-water 1:1 (υ/υ ) as mobile phase. The spots were visualized under UV light. The effect of impurities (metal cations and inorganic anions) on the chromatography of thiamine hydrochloride was examined. The detection limit for thiamine hydrochloride was 0.09 µg per spot and the relative standard deviation of the R F value of thiamine hydrochloride in five analyses was 14.99%. The applicability of the method to the identification of thiamine in pharmaceutical formulations was also tested.