The present study aimed to study the distribution of single nucleotide polymorphisms in toll-like receptor 2 (TLR2) gene in two important cattle breeds Friesian (locally adopted) and Bhagnari (indigenous) of Pakistan. TLR2 is a key receptor in the innate immune system by recognizing a variety of microbial ligands known as pathogen associated molecular patterns (PAMPs). To date, ten TLRs (TLR1 to TLR10) have been discovered in cattle. In this research work, DNA samples of the included breeds were provided by Molecular Biology and Biotechnology laboratories, Virtual University of Pakistan. The DNA samples were quantified and used for PCR amplification through six pairs of primers enclosed complete gene. The complete bovine TLR2 gene showed two exons with CDS of 2355 bp encoding 784 aa long protein with predicted molecular weight 104 kDa and 6.97 pI value. Twenty samples of each breed were amplified and sequenced for mutational analysis. A total of nineteen (19) polymorphisms were detected in the CDS of TLR2 gene in Friesian cattle. Among them six polymorphisms were non-synonymous at positions p.63E>D, p.326Q>H, p.337K>R, p.417S>N, p.563H>R, p.665Q>H and thirteen polymorphisms were synonymous. Thirteen were transition type six were transversion type mutation. One polymorphism was identified as novel and rest were reported in earliest studies. Eleven variations were observed in the ECD, two in transmembrane and five were detected in TIR domain. A total of seven polymorphisms were detected in the CDS of TLR2 in Bhagnari cattle. Five polymorphisms were non-synonymous at positions p.147Q>P, p.227L>F, p.335I>T, p.345S>N and p.605M>T and two polymorphisms were synonymous. Five polymorphisms were transition type and two were transversion type mutations. Three of them were identified as novel variations. Four polymorphisms were observed in the ECD, one in transmembrane and two were TIR domain. SNPs identified in EC domain in both breeds fell within the leucine-rich repeats (LRR) region that responsible for ligand recognition. The ratio of dS/dN substitutions was <1 at polymorphic-sites indicating purifying selection. The deduced amino acid sequence revealed a signal peptide (20 amino acids) conserved EC domain (54-584 amino acids) with 20 motifs of leucine rich repeats (LRR), transmembrane domain (585-607 amino acids) and Toll-IL receptor domain (633-783 amino acids). The 3D structure of TLR2 in both breeds is to be solenoid structure based on the positions of LRR. Phylogenetic analysis was performed revealed clustering of Bhagnari with Bos indicus and Friesian with Bos taurus as the nearest neighbor. The polymorphisms in TLR2 can be useful in future research exploring its role in immunity and may use as a marker for disease resistance by selective breeding.