The variability of disease resistance genes in traditional breeds is considered to be a reservoir of functional variants for breeding programs. The population equilibrium is supposed to reflect local infection pressure while the rare variants are useful in counteracting the gene pool erosion. Screening for the diversity of innate immunity genes has been performed in two conserved breeds of cattle, Czech Red and Czech Red Pied. The survey comprised ten members of bovine TLR gene family coding for Toll-like receptors involved in early pathogen recognition. Population of 115 animals included most of living individuals of each breed and archived samples. Along with Sanger sequencing, polymorphism has been discovered in pooled amplicons using the PacBio NGS platform and subsequently validated with designed genotyping assays. An independent pipeline for processing long PacBio reads has been developed which combined primary data quality check with FastQC, assembly with Ugene and removal of duplicate PCR with Picard Tools followed by variant calling using SAMtools and filtration with VCFtools. The polymorphism revealed in a small population of historical breeds was unexpectedly high. The numbers of detected polymorphisms and reconstructed haplotypes (in brackets) in the antibacterial series were 6 (4) in TLR1, 24 (6) in TLR2, 8 (18) in TLR4, 26 (6) in TLR5, and 4 (6) in TLR6, while the mostly antiviral TLR3, TLR7, TLR8, TLR9, and TLR10 harbored another 70 SNPs and indels. The observed diversity approaches general diversity in TLRs as reported for the panel of world breeds (Fisher et al., Plos One 6:11, 2011). Breed specificity can be exemplified in TLR4 where three haplotypes were shared in similar frequencies while the other haplotypes showed preferences for one of the local breeds. Namely, haplotype B1, otherwise common in European breeds, was greatly reduced in Czech Red.