Diacylglycerol aminotransferase (DGAT1) gene present in the QTL region is one of the potential candidate genes that have significant effect on milk fat content in dairy cattle. An alanine to lysine substitution (K232A) due to dinucleotide substitution in exon-8 of DGAT1 has been identified to be responsible for milk fat variation in different bovine breeds. The present study was carried out in Sahiwal milking cows to characterize the DGAT1 gene through polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) genotyping. Whole blood sample (3 ml) was collected from 70 unrelated milking Sahiwal cattle maintained at Livestock Research Station, Kodamdesar, Bikaner, Rajasthan in an aseptic manner. Genomic DNA was extracted from whole blood through spin column method. The quality and concentration of extracted DNA was checked on 0.8% agarose gel and Nano Drop spectrophotometer, respectively. The exon 8 region of DGAT1 gene was amplified using species specific primers (GenBank Accession No. AJ318490). The amplified 411bp region of DGAT1 gene was digested overnight with 5U of Eae I restriction enzyme in a mixture containing 10X buffer and nuclease free water. The genetic variability in exon 8 of DGAT1 gene was assessed on 8% polyacrylamide gel electrophoresis. The frequency of different alleles and genotypes of DGAT1 gene were estimated through standard procedure of Falcon and Mackay (1998). All the analyzed samples showed ‘KK’ genotype with single fragment of 411bp and the gene and genotype frequency were observed fixed. The study concluded that exon 8 of DGAT1 gene was monomorphic in Sahiwal cattle for K232A region and other single nucleotide polymorphisms (SNP’s) of the region should be explored to detect individual variation in milk fat yield. [ABSTRACT FROM AUTHOR]