A production herd of Czech Simmental cattle (Czech Red Pied, CRP), the conserved subpopulation of this breed, and the ancient local breed Czech Red cattle (CR) were screened for diversity in the antibacterial toll-like receptors (TLRs), which are members of the innate immune system. Polymerase chain reaction (PCR) amplicons of TLR1, TLR2, TLR4, TLR5, and TLR6 from pooled DNA samples were sequenced with PacBio technology, with 3–5_coverage per gene per animal. To increase the reliability of variant detection, the gDNA pools were sequenced in parallel with the Illumina X-ten platform at low coverage (60_ per gene). The diversity in conserved CRP and CR was similar to the diversity in conserved and modern CRP, representing 76.4% and 70.9% of its variants, respectively. Sixty-eight (54.4 %) polymorphisms in the five TLR genes were shared by the two breeds, whereas 38 (30.4 %) were specific to the production herd of CRP; 4 (3.2 %) were specific to the broad CRP population; 7 (5.6 %) were present in both conserved populations; 5 (4.0 %) were present solely for the conserved CRP; and 3 (2.4 %) were restricted to CR. Consequently, gene pool erosion related to intensive breeding did not occur in Czech Simmental cattle. Similarly, no considerable consequences were found from known bottlenecks in the history of Czech Red cattle. On the other hand, the distinctness of the conserved populations and their potential for resistance breeding were only moderate. This relationship might be transferable to other non-abundant historical cattle breeds that are conserved as genetic resources. The estimates of polymorphism impact using Variant Effect Predictor and SIFT software tools allowed for the identification of candidate single-nucleotide polymorphisms (SNPs) for association studies related to infection resistance and targeted breeding. Knowledge of TLR-gene diversity present in Czech Simmental populations may aid in the potential transfer of variant characteristics from other breeds.