Objective To investigate the distribution characters and linkage disequilibrium of estrogen receptor gene PvuⅡ, XbaⅠ and Codon325 polymorphisms. Methods The single nucleotide polymorphisms (SNPs) of estrogen receptor gene of 678 normal male subjects from Southern Yangtse River were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time fluorescence quantitative PCR. All the subjects were from cities of Jiangsu and Zhejiang provinces, and from Yueyang, Hunan Province. Results According to the different geographical distribution, subjects were divided into two groups: group of Jiangsu and Zhejiang Provinces and group of Yueyang, Hunan Province. The genotype frequency of the two groups reached Hardy-Weinberg equilibrium. Moreover, the frequency distribution of PvuⅡ and Codon325 loci was different between the two groups (P<0.05). In addition, strong linkage disequilibrium was only found between PvuⅡ and XbaⅠ, neither between PvuⅡ and Codon325 nor between XbaⅠ and Codon325. Conclusions The frequency distribution of SNPs of estrogen receptor gene exists regional difference. There is an apparent linkage disequilibrium between PvuⅡ and XbaⅠ loci of estrogen receptor gene in men of Southern Yangtse River.