The variants of the genes coding for the Toll-like receptors 1, 2, 4, 5 and 6 with anti-bacterial function were tested for the association with milk utility, udder health and reproductive traits in Czech Red Pied (Czech Simmental) cattle population. Data from amplicon resequencing with PacBio technology and from the whole-genome resequencing with HiSeq X-Ten technology complemented each other according to the principle of hybrid sequencing, which increases the reliability of variant calling. A set of 150 bulls was genotyped with primer extension technique for 36 polymorphisms present. The effect of genotype classes was tested against 15 phenotypic traits with one-factor ANOVA. The effect on the milk utility traits was exerted mostly by the variants in TLR2 and TLR5, partially also in TLR1 and TLR6, but no effect was produced by the TLR4 variation. As expected, the TLR1 and TLR6 variants affected udder health traits. In addition, unexpected associations of the variants in TLR1, TLR2 and TLR6 with cystic ovary incidence, early reproductive disorders and calving ease were observed. The non-immune functions of TLRs were suggested as the conceivable mechanism of the action on reproductive traits, along with the effect mediated by infection incidence. The observation was related to the previously reported quantitative trait loci for calving ease. In addition, variants in TLR4 were associated with calf vitality index, which is consistent with the increased role of infections compared to the adult animals.