Litter size is one of the most important economic traits in pig production, and the more piglet numbers per litter is capable to increase pork production and bring more economic profit for pig industry. ESR (estrogen receptor) gene has been determined to be one of the major genes affecting phenotype of litter size without any genetic negative correlation to growth and carcass traits. An optimized standard PCR-RFLP protocol is employed to type 262 sows from 5 different breeds in ESR loci, and then with the computation based on linear model ESR gene is confirmed to be a major locus significantly associated with litter size (P < 0.001). The genetic effect of ESR gene is quite large in these breeds, especially in these Chinese pig population. The sows of beneficial homozygote BB produce 1.40-3.37 total number born/litter and 0.63-3.58 number born alive/litter more than the sows of non-beneficial homozygote AA do. The information found in the present study is very important and could be utilized as DNA marker for improvement of reproduction trait in practice of pig breeding.