The application of the developed approach for the screening polymorphism of TLR genes in a representative group of historical and modern cattle breeds from Russia is relevant today. The pipeline included the stages of obtaining overlapping amplification products from the coding regions of all ten cattle TLR genes, their subsequent purification and normalization. While the antibacterial group included TLR1, TLR2, TLR4, TLR5, and TLR6, the antiviral group includes TLR3, TLR7, TLR8, TLR9, and TLR10 (despite unclear specificity). For the analysis, 575 animals were used, both bulls and cows. Combined samples containing equimolar concentrations of amplicons obtained from pooled genomic DNA were sequenced on the PacBio platform. Identified structural 36 variants of TLR were annotated according to their biological significance. Both new and already identified patches of variability, already annotated and documented in dbSNP, were found. The data are necessary for the further breeding of local breeds in Russia with regard to their natural resistance to various diseases.