The aim of present study was to screen for the presence of genetic variability in thioesterase (TE) domain of the fatty acid synthase (FASN) gene in Murrah buffalo, an important milch breed of India. TE domain of FASN gene is responsible for termination of fatty acid chain during its de-novo synthesis which in turn affects the fatty acid composition and quality of milk fat. In this study, terminal exons 38 to 42, including TE domain were explored for the presence of SNP variations using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. An important SNP at g.18433A>G in exon-40 region of FASN gene was identified in Murrah buffaloes. Three types of genotypes, viz. AA, AG and GG were observed having 34%, 56% and 10% frequencies, respectively. The allele frequencies of A and G alleles were 0.62 and 0.38, respectively. The identified polymorphism was non-synonymous transition in FASN gene. The study will augment the information available and can be applied in future studies to determine the role of buffalo FASN gene as a candidate gene marker for a milk-fat content.