Toll-like receptors (TLRs) are a family of type I transmembrane pattern recognition receptors (PRRs) that play a critical role in the innate immune system. They recognize specific molecular patterns from a wide variety of pathogens and initiate a signaling cascade that mobilizes the appropriate host defense. They are usually expressed on membrane of various subclasses of immune and non-immune cells such as macrophages, monocytes, dendritic cells, neutrophils, T-cells, B-cells, epithelial, endothelial and fibroblasts. In the present study, the genetic polymorphisms in Toll-like receptors 2 (TLR2) gene was investigated in indigenous cattle breeds Achai and Lohani of Khyber Pakhtoon Khawa (KPK) province of Pakistan. The complete sequences of TLR2 gene composed of 5?UTR 136bp, coding sequence 2355bp and 3?UTR 1316 bp. The distribution of polymorphisms was explored in the coding region of TLR2 gene in studied two cattle breeds. The phylogenetic analysis was performed on the basis of coding region of TLR2 gene showed that the Bos indicus, Bos taurus as the nearest neighbors with other species bubalus bubalis, Bos frontalis, Bos grunniens, and Bison bison. The Ovis aries and Capra hircus in a separate group with Capra ibex, Antidorcas marsupialis, Damaliscus pygargus and the chicken (Gallus gallus) was found to be the farthest specie. The ratio of dS/dN substitutions was <1 at polymorphic-sites indicating purifying selection. A total of seven polymorphisms were detected in Achai cattle breed, in which two variations were synonymous and five mutations were non-synonymous. In the analysis, four polymorphisms were transitions type mutations and three were transversion. The amino acid [aa] sequences analysis of the coding region (784 aa) revealed extracellular domain composed of 20 leucine-rich repeats (LRRs), trans membrane and Toll-IL receptor domains. A total of four mutations were observed in Extracellular domain, one in transmembrane domain, and two in TIR domain of TLR-2 protein in Achai cattle breed. The Lohani cattle breed was more polymorphic at TLR2 gene and a total of 41 polymorphisms were detected including 20 variations were synonymous and 21 were identified non-synonymous. Similarly, the amino acid [aa] sequences analysis of the coding region (784 aa) revealed 12 extracellular domain composed of 20 LRRs, trans membrane and Toll-IL receptor domains. A total of 25 polymorphisms were observed in the Extracellular domain, four in transmembrane domain and five in TIR domain, one in signal peptide. The predicted bovine TLR2 is a succulent like having energetic sites buried inside the concave side and forming a pocket for ligand-binding. The present study findings show that the variation may be associated with pathogens associated molecular pattern (PAMPs) mediated differential TLR-signaling of bovine immune response and could regulate the consequence of infection