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Background: Triacylglycerols are quantitatively most important storage form of energy for eukaryotic cells. Acyl CoA: Diacylglycerol O-acyltransferase 1 (DGAT1, EC 2.3.1.20) is a microsomal enzyme that catalyzes the terminal and only committed step in triacylglycerol synthesis by using diacylglycerol and fatty acyl CoA as substrates. Objective: The present study was undertaken with the objectives to study the molecular differences in DGAT1 among different ruminants. Materials and Methods: The DGAT1 was partially amplified (411 bp) by designing gene specific primers and confirmed by sequencing the amplicon and its comparison with DGAT1 gene of other ruminants (i.e., bovine, buffalo and goat). Results: Comparative study of DGAT1 among different ruminants reveals different level of mutation with respect to its gene sequence (0.0-0.4%) and protein sequence (0.0-1.2%). Conclusion: Molecular differentiation among different ruminants reveals that bovine and buffalo are very close to each other and sheep is related to goat. Further study is underway to detect polymorphism and associated them with milk fat trait in sheep.