Regulatory region of milk protein alpha S2-casein (αS2-CN) gene sequence was characterized and analyzed for nucleotide variations in animals representing 13 Indian zebu cattle (Bos indicus) breeds. A total of 15 variations; 11 in promoter region (1.56 Kb): -1481 (C>T), -1412 (C>T), -1342 (C>T), -1084 (G>A), -979 (A>G), -657 (A>T), -508 (A>G), -186 (T>C), -184 (T>C), -151 (T>C) and -135 (C>T); 1 in 5'-UTR (44 bp): 7 (C>T) while, 3 in intron-I region (73 bp): 186 (C>T), 194 (A>C) and 301 (A>T) were identified. Additionally, single deletion was observed at -975 (A>-) but not involve any known potential transcription factor binding sites (TFBS). Comparison with Bos taurus sequence revealed two additional variations -1085 (T>C) and -739 (A>G). Out of the total 18 variations observed between indicine and taurine αS2-CN regulatory region sequence, 15 were novel to B. indicus and are reported for the first time. Among these, four variations were located within the potential TFBSs; -1342 (C>T) within HNF-3beta, -739 (A>G) within C/EBP-alpha while -657 (A>T) and -508 (A>G) were found within glucocorticoid receptor TFBSs. Variations located within or in proximity to putative TFBSs could possibly influence the binding affinity of nuclear factors towards DNA binding domains, thus affecting transcriptional rate of αS2-CN gene. Phylogenetically, as expected, Indian zebu cattle were grouped close to B. taurus and were most distantly placed in comparison to human. The study indicated possible genetic variations in the regulatory regions of αS2-CN gene within Indian native cattle (B. indicus) and also its comparison with evolutionary different B. taurus breeds.