DGAT 1 enzyme catalyses the final step of milk triglyceride biosynthesis. A lysine to alanine substitution (K232A) at nucleotide position 10433-10434 at DGAT 1 is significantly associated with milk production traits. This polymorphism may potentially be used as a marker in selection programmes to enhance the production potential and to accelerate the rate of genetic gain. In the present investigation, DGAT1 K232A polymorphism was analysed in crossbred Hardhenu (HF X SW X Haryana) cattle (n=50) by restriction digestion of a 413 bp PCR fragment by Eae I. An undigested fragment of 413 bp indicated the K allele whereas two co-migrated fragments of 210 and 203 bp length indicated the A allele. In Hardhenu cattle population genotype frequencies were 0.30 (KK) and 0.70 (AK). The frequency of K and A allele were 0.65 & 0.35. Over all 15 KK and 35 AK animals were recognised in Hardhenu cattle whereas no animal of AA genotype were observed. The overall DGAT1K allele frequency in crossbred cattle was 0.65.