Toll-like receptors play role in the innate immune responses and orchestrate the adaptive immunity by induction of proinflammatory cytokines and up-regulation of costimulatory molecules. The present study has characterized TLR2 cDNA in nilgai, buffalo, sheep and goat that recognizes the peptidoglycan of Gram-positive bacteria. TLR2 coding sequences were amplified from monocytes cDNA and cloned in pGEMT-easy vector for nucleotide sequencing. Sequence analysis revealed 2355-bp-long TLR2 open reading frame encoding 784 amino acids in all the species studied. Nilgai TLR2 has 97.8% to 95.1% identity at nucleotide level and 96.2% to 92.7% identity at amino acid level with other ruminant species studied. Nonsynonymous substitutions exceeding synonymous substitutions indicated evolution of this receptor through positive selection among ruminants. Furthermore, basal TLR2 messenger RNA expression in different immune cells and tissues quantified by real-time polymerase chain reaction revealed highest level in in vitro derived dendritic cells followed by peripheral blood mononuclear cells. Skin and testes also expressed significant level in both nilgai and buffalo. Comparatively, nilgai immune cells and tissues expressed more TLR2 transcript than buffalo, thus elucidating stronger armamentarium of antibacterial immunity in nilgai as compared to buffalo.