3435C>T, a single nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene, is linked to the variability of P-gp expression and function among different individuals. It was found that ethnic differences exist in the polymorphism of the MDR1(C3435T) gene. However, the distribution of 3435C>T genotypes in the Chinese Han population is not clear up till now. In this study, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was applied to assess 3435C>T genotypes in 265 healthy individuals of the Han population of China. The genotype frequencies were: CC 32% (n = 85), CT 48% (n = 127) and TT 20% (n = 53). C and T allele frequencies were 0.56 and 0.44 respectively. The C allelic frequency for female was 56.5% (T: 43.5%), and 55.9% (T: 44.1%) for male. There was no significant difference between females and males (p = 0.92, OR = 1.02, 95%CI: 0.67-1.57) in the present study. The frequency of C-allele was similar to that of some populations in Asia/Europe and was lower than that of populations in Africa. When compared with the study on Singapore-Chinese, some differences were found. The results of this study would be useful for individualised therapy of some diseases, and could have a prognostic implication for the Chinese Han population.