This experiment was carried out to determine the evolution of the TLR3 gene in Nigerian indigenous and exotic turkeys. A total of 150 turkeys i.e. 100 exotic and 50 indigenous were used for this study. Birds were allotted treatments and fed formulated grower diets that meet the NRC—Turkey grower nutritional requirements. Genomic DNA was isolated from each blood sample, and the concentration and purity of each DNA sample was determined using a Nanodrop Spectrophotometer. After DNA extraction and quantification, three DNA samples from each genetic group (or breed) were taken for TLR3 gene discovery and polymerase chain reaction (PCR). The source sequence and the retrieved sequences were trimmed and edited while the amino acid sequences of the new turkey TLRs were predicted based on the open reading frames of the expressed nucleotide sequences, and the nomenclatures of turkey TLRs were based on the best hits of the proteins in the database. From the findings of the study, the SNP (172N>N) detected at position 516 was synonymous in both exotic and local turkey breeds, and this resulted in amino acid changes from Alanine to Threonine in exotic turkeys and Cysteine to Tyrosine in local turkeys. Also, in this study, the SNP (7 Q>*) detected in exon 4 was non-synonymous, resulting in amino acid changes from Cytosine to Threonine in the exotic turkey. Positive Tajima's D values were the same for exotic and local turkey breeds. The findings of this study suggest significant instances of purifying selection acting on the gene. Based on the results of this research, the populations of local and exotic turkeys showed a low level of heterozygosity at TLR3 locus and may be homogenous. Also, there was low genetic diversity in the TLR3 gene of both local and exotic turkey breeds. This suggests potential vulnerabilities and challenges for their long-term health and adaptability. Strategies that aim to increase genetic diversity, like cross-breeding should be implemented.