This Research Communication describes the polymorphisms in the coding region of DGAT1 gene in Riverine buffalo, Swamp buffalo and crossbred buffalo, and associations between polymorphisms and milk production performance in Riverine buffalo. Two polymorphisms of DGAT1were identified, located in exon 13 and exon 17, respectively. The distribution of the genotypes of the two SNP loci in different buffalo population varied, especially the polymorphism located in exon 13 which was not found in the Swamp buffalo. Moreover, SNP located in exon 17 was a nonsynonymous switch resulting in the animo acid sequence changed from an arginine (Arg) to a histidine (His) at position 484. Both SNPs were in Hardy–Weinberg equilibrium, and the polymorphism of g.8330T>C in the exon 13 was significantly associated with peak milk yield, total milk yield and protein percentage. The C variant was associated with an increase in milk yield and peak yield but less in protein percentage compared with the T variant. The polymorphisms of g.9046T>C in exon 17 were significantly associated with fat percentage, in that the buffaloes with TT genotype had a significantly higher fat percentage than those with CC genotype. These findings reveal the difference in the genetic evolution of the DGAT1 between Riverine buffalo and Swamp buffalo, and provide evidence that the DGAT1 gene has potential effects for Riverine buffalo milk production traits, which can be used as a candidate gene for marker-assisted selection in buffalo breeding.