Alpha-lactalbumin (alpha-LA) is a major whey protein found in milk. Polymorphs of alpha-LA gene are reported to be significantly associated with milk production and constituent traits. Therefore, the present study was undertaken to detect polymorphism in alpha-LA at the genic level and to explore allelic variability at this locus. A total of 196 animals, belonging to four breeds of riverine buffalo viz. Bhadwari, Mehsana, Surti and Murrah were included under the present investigation. Two fragments i.e. 133 bp (Exon 1) and 159 bp (Exon 2) of alpha-LA gene were amplified by polymerase chain reaction and subsequently, single strand confirmation polymorphism (SSCP) study was carried out to identify different allelic pattern and genotypes of the animal included in the study. Both fragment of alpha-LA gene was found to be polymorphic in all the four breeds of riverine buffalo. Number of genotypes and allele varied breed to breed for both the fragments. In case of 133 bp fragment, four alleles A, B, C and D were found among different breeds of buffalo whereas in 159 bp fragment, five alleles namely A, B, C, D and E was found in different breeds. Nucleotide sequence data of different alleles showed the presence of both silent as well as functional mutation leading to variability in polypeptide chain.