The haplotype structure of anti-bacterial Toll-like receptor genes (TLR1, -2, -4, -5 and -6) in Czech Red Pied cattle was explored. Initially, single nucleotide polymorphisms were detected by hybrid resequencing that combined PacBio RSII and WGS with HiSeq X Ten. 15 haplotypes were directly revealed in TLR2 by amplicon sequences and two distinct holotype groups were detected using the Network program. Clustering in TLR2 was independently confirmed for statistically reconstructed haplotypes. The bimodal distribution was also detectable in the haplotypes of TLR5 and -6, while only a trend was seen in TLR4 and -1.