Abstract: The prolactin exerts obvious adjustment and control function for mammary gland development, lactation and milk protein gene expression in water buffalo. In this study the sequence features and polymorphisms of the exon 4 in prolactin gene were examined in 385 individuals which came from 12 river and swamp type buffalo populations using DNA direct sequencing and PCR-SSCP methods. The results showed that the sequence of exon 4 in prolactin gene was consists of 180 nucleotides, the fragment had high conservative character in different species. The e4. 109 C＞T substitution was detected in nine swamp buffalo populations, and it was a silent mutation and was not associated with the traits of milk yield in buffalo. The PB^A gene was the predominant gene in seven swamp type buffalo populations, while PB^B gene was the dominant gene in Dehong and Fuzhong populations. The frequencies of PB^A in swamp type buffalo was 0.400 − 0.917 and the average value was 0.629±0.049. The polymorphism wasn’t found in river buffalo, all the samples from river buffalo were holding nucleotides e4.109 C. The results indicate that there is distinct genetic differentiation between swamp and river type buffalo.