Abstract: The study of functional gene variations in Indigenous cow breeds is scarce, with only a few breeds being investigated using PCR-RFLP. In the Maharashtra state of India, remote villages were visited to collect 32 samples of cattle belonging to eight breeds as part of this study. By resequencing the 403 bp of the Exon IV CSN3 allele, haplotypes were derived. Fourteen genotypes (G1 to G14) defined by seven SNPs were found in a small panel of thirty-two samples. Three haplotypes (H01, H02, and H03) were reconstructed from these 14 genotypes, and their ratio were calculated. Two of the three haplotypes (H01, H03) match the CSN3*B4, CSN3*B, CSN3*H, and CSN3*G alleles, while the third haplotype (H02) was discovered to be a novel allele because it varies from the CSN3*B4, CSN3*B, and CSN3*H, CSN3*G alleles by mutation at location C5306T-Ile100Thr. The initial report in this study cluster cited three haplotypes, which contained A1, B2, B4, B, G, and H alleles that were previously documented in both subspecies of Bos taurus, The Neighbor Joining (NJ) tree building shows that there might be some sharing of genetic traits or mixing between Bos taurus indicus and Bos taurus taurus. Finding a new allele highlights the importance of looking at local breeds for different gene traits that can have significant economic benefits to stop the loss of genetic quality, this project was designed to record the native cow breeds.