The Sox gene family constitutes transcription factors with a conserved high mobility group box (HMG) that regulate a variety of developmental processes, including sex differentiation, neural, cartilage, and early embryonic development. In this study, we systematically analyzed and characterized the 20 Sox genes from the whole buffalo genome, using comparative genomic and evolutionary analyses. All the buffalo Sox genes were divided into nine sub-groups, and each gene had a specific number of exons and introns, which contributed to different gene structures. Molecular phylogeny revealed more sequence similarity of buffalo Sox genes with those of cattle. Furthermore, evolutionary analysis revealed that the HMG domain remained conserved in the all members of the Sox gene family. Similarly, all the genes are under strong purifying selection pressure; seven segmental duplications occurred from 9.65 to 21.41 million years ago (MYA), and four potential recombination breakpoints were also predicted. Mutational analysis revealed twenty non-synonymous mutations with potential effects on physiological functions, including embryonic development and cell differentiation in the buffalo. The present study provides insights into the genetic architecture of the Sox gene family in buffalo, highlights the significance of mutations, and provides their potential utility for marker-assisted selection for targeted genetic improvement in buffalo.