Abstract Previous studies have reported that a non-conservative substitution of lysine by alanine (K232A) in the 8th exon of acyl CoA:diacylglycerol acyltransferase (DGAT1) gene in cattle has a major effect on milk composition and yield. But yet little research has been utilized in this gene segment in buffalo. In this study the genetic differentiation of three indigenous Anatolian buffalo populations has been investigated in the 8th exon of DGAT1 gene. Fourteen out of the 24 restriction enzymes have a recognition site on the DGAT1 gene segment, generating a total of 26 restriction sites. AluI, HincII and HphI restriction enzymes out of these 14 enzymes were found to detect polymorphism. K232A substitution found in cattle breeds with CfrI restriction enzyme is not a diagnostic site in Anatolian buffalo. Only lysine variant (K allele) is found in all of the Anatolian buffalo tested. In this study, four different haplotypes were obtained and sequencing of this gene revealed three polymorphic nucleotide substitutions in the 8th exon of the buffalo DGAT1 gene. C→T, A→G and C→G base substitutions at positions 43, 154 and 373, respectively, each generated two different fragment patterns in Anatolian buffalo. Type 1 haplotype was the most common haplotype, found in 73.2% of the samples. Along with the polymorphic substitution sites in DGAT1 gene, this study provides evidence that all the Anatolian buffalo have fixed allele with respect to DGAT1 K allele reported to be responsible for high milk fat yield. Our study presents the first comprehensive sequencing analysis of Anatolian buffalo and it is the first time that sequencing data from DGAT1 gene segment have been obtained at the population level.