Thiamine status in response to a wide range (2 to 32 mg/kg) of dietary supplementation of thiamine was studied in male and female broiler chickens in order to understand the manifestation of marginal thiamine deficiency in chickens. Thiamine content and transketolase (TK) activity, were measured in tissues (brain, heart, liver) and blood samples from chickens at 1, 7, 14, 21, 35, and 42 days of age. There was no correlation between body weights and tissue thiamine or TK activity at 3 or 6 weeks of age. Overall thiamine supplementation did not have significant effects on TK activity in the liver (p = 0.22) or brain (p = 0.07). Males had a higher concentration of thiamine than females (p < 0.001) in livers and brains, but the level in the heart did not differ significantly between sexes. There were substantial contrasts among tissues in changes in the tissue thiamine content in response to dietary thiamine levels. Relative to day 1, unsupplemented birds' heart, brain, and liver thiamine increased by 34, 30, and 31% respectively. In response to supplementation, the heart tissue tended to accumulate thiamine at a considerably higher rate than liver or brain. Blood thiamine concentration tended to decline in unsupplemented birds and those supplemented at 2 and 4 mg/kg levels, whereas in birds supplemented with 8 mg blood thiamine level increased at day 7 and remained relatively constant for the remaining part of the trial. Supplementation at the levels of 16 and 32 mg/kg resulted in blood thiamine increasing throughout the duration of the trial. The presented data indicate that there are organ specific differences in the requirement for thiamine. The heart appears to have a higher requirement than the liver or brain. Since there are potential beneficial effects of thiamine on the heart, this subject deserves more detailed investigation.