Regulatory region of milk protein alpha S1-casein (αS1-CN) gene was sequenced, characterized, and analyzed to detect variations among 13 Indian cattle (Bos indicus) breeds. Comparative analysis of 1,587 bp region comprising promoter (1,418 bp), exon-I (53 bp), and partial intron-I (116 bp) revealed 35 nucleotide substitutions (32 within promoter region, 1 in exon-I, and 2 in partial intron-I region) and 4 Indels. Within promoter, 15 variations at positions −1399 (A > G), −1288 (G > A), −1259 (T > C), −1158 (T > C), −1016 (A > T), −941 (T > G), −778 (C > T), −610 (G > A), −536 (A > G), −521 (A > G), −330 (A > C), −214 (A > G), −205 (A > T), −206 (C > A), and −175 (A > G) were located within the potential transcription factor binding sites (TFBSs), namely, NF-κE1/c-Myc, GATA-1, GATA-1/NF-E, Oct-1/POU3F2, MEF-2/YY1, GATA-1, AP-1, POU1F1a/GR, TMF, GAL4, YY1/Oct-1, HNF-1, GRalpha/AR, GRalpha/AR, and AP-1, respectively. Seventy-four percent (26/35) of the observed SNPs were novel to Indian cattle and 11 of these novel SNPs were located within one or more TFBSs. Collectively, these might influence the binding affinity towards their respective nuclear TFs thus modulating the level of transcripts in milk and affecting overall protein composition. The study provides information on several distinct variations across indicine and taurine αS1-CN regulatory domains.