We report molecular cloning and single nucleotide polymorphism detection of the buffalo DGAT1 gene. Diacylglycerol acyltransferase (DGAT1) is considered the key enzyme in controlling the rate of synthesis of triglycerides. The DGAT1 gene was recently identified as a strong functional candidate gene affecting milk yield and composition in cattle. A full-length buffalo DGAT1 genomic DNA was amplified by iterative PCR based on homolog cloning. The buffalo DGAT1 gene comprises 17 exons and spans approximately 8.3 kb. The genomic structures of DGAT1 are highly conserved among mammal species. The deduced protein of buffalo DGAT1 contains 489 amino acids, showing high-sequence similarity with mammal homologs. Through PCR-SSCP analysis and sequencing, seven polymorphic positions were detected in the complete genomic region of buffalo DGAT1, and their frequencies were observed from a collection of 117 buffalo. The SNP (C/T) detected at position 11785 in exon 17 creates a substitution change for the amino acid sequence, resulting in an Ala residue (GCG) transition to a Val residue (GTG) in position 484 of buffalo DGAT1 protein. Information provided in this study will be useful in further studies to determine the role DGAT1 plays in the regulation of milk fat synthesis and quality improvement for milk in buffalo.