Alpha S1-casein (αS1-CN) is a major casein in milk, which exerts a crucial role in casein transport and is related to individual milk components, nutritive value and production traits of milk. So far, αS1-CN coding gene (CSN1S1) has been widely studied in dairy cattle, but the polymorphisms of the CSN1S1 gene have not been fully understood in buffalo. In this study, the polymorphisms in coding sequence (CDS) of the CSN1S1 gene for river and swamp buffalo were detected using PCR product direct sequencing. The CDS for both types of buffalo was the same in length, which contained 645 nucleotides and encoded a peptide composing of 214 residues. A total of 5 single nucleotide polymorphisms (SNPs) was identified in two types of buffalo. Among them, c.516T>C and c.578C>T were observed only in river buffalo, while c.175A>G, c.580T>C and c.609T>G were found only in swamp buffalo. The c.175A>G, c.578C>T and c.580T>C were non-synonymous, which led to substitutions of p.I44V, p.L178S and p.F179L. The prediction showed that the p.F179L may affect the function of buffalo αS1-CN. Eight buffalo CSN1S1 haplotypes were defined in this study, and accordingly, 6 protein variants and 2 synonymous variants of αS1-CN were inferred and named. The variants A, B’, B’’, C, E and F were observed only in river buffalo, whereas variant D was found only in swamp buffalo. The variant B was shared by both types of buffalo with high frequencies. The buffalo variants determined here did not exist in Bos genus. In addition, 9 amino acid differential sites of αS1-CN between buffalo and Bos genus were identified, of which p.42T and p.115S were located at phosphorylation sites, which may lead to differences in the physicochemical properties of αS1-CN between buffalo and Bos genus.