Indian milch bovines comprise 58.56% of the total livestock popula on (512.05 million) in the country and primarily include na ve and crossbred ca le (37.28%) and water buffaloes (21.28%). Milk and milk products are essen al food items in Indians' diet, especially in children and the elderly and senile. Milk fat is an important cons tuent of milk and has an economic value and its percentage in milk varies between species and breeds within species. Butyrophilin (BTN1A1) is a membrane protein that regulates the secre on of lipids and size of a fat globule in milk. The present study was conducted on 538 bovines from 11 breeds/popula ons adapted to different parts of India, with an aim to screen and determine the major allele of the BTN1A1 gene. The genotyping of samples was done using PCR-RFLP based tests. The results indicated that exon8 of the BTN1A1 gene was polymorphic in the Tharparkar, Sahiwal, Jhari, and Belahi popula ons of the na ve ca le, as well as the Holstein Friesian and Jersey crossbreeds. Meanwhile, exon 8 was monomorphic in the Murrah, Chilika, Gojri, Chha sgarhi, and Bargur popula ons of water buffalo. Allele A was iden fied as the major allele in Indian bovines. We conclude that varia ons in the BTN1A1 gene can serve as an excellent gene c marker in the selec on of ca le with higher milk fat, and can be applied when formula ng their breeding plans.