The aim of the present study was to investigate the variability at CSN1S1 locus of the Italian Mediterranean river buffalo and to study possible allele effects on milk yield and its composition. Effects of parity, calving season and month of production were also evaluated. Three single-nucleotide polymorphisms were detected. The first mutation, located at position 89 of the 17th exon (c.628C>T), is responsible for the amino acid change p.Ser178 (B allele)/Leu178 (A allele). The other two polymorphisms, detected at the positions 144 (c.882G>A) and 239 (c.977A>G) of 19th exon, respectively, are silent (3' UTR, untranslated region). Associations between the CSN1S1 genotypes and milk production traits were investigated using 4122 test day records of 503 lactations from 175 buffalo cows. Milk yield, fat and protein percentages were analysed using a mixed linear model. A significant association between the c.628C>T SNP and the protein percentage was found. In particular, the CC genotype showed an average value ~0.04% higher than the CT and TT genotypes. The allele substitution effect of cytosine into thymine was –0.014, with a quite low (0.3%) protein percentage contribution to total phenotypic variance. A large dominance effect was detected. Characterisation of the CSN1S1 transcripts and a method based on MboI amplification created restriction site PCR for a rapid genotyping of c.628C>T are provided.