Screening for polymorphism in the anti-bacterial TLR genes in the Czech Simmental cattle population was performed by hybrid resequencing of pooled samples using WGS with Hiseq X-Ten technology and amplicon panel with PacBio RSII. Subsequently, polymorphisms were genotyped in 164 bulls with primer extension assays. Associations with combined phenotypic traits comprising four udder health indicators, five milk production and six reproductive traits, including calving ease, indicated the effects of TLR1, -2 and -6 on reproductive traits. The shared effect pattern can be explained by the interaction of the products in TLR1/TLR2 and TLR6/TLR2 heterodimers. The TLR4 and TLR5 variants were associated with changes in reproductive traits as well. In addition, the TLR5 polymorphism was associated with milk production traits. No effect on udder health was observed in TLR4 and TLR5, in contrast to the expectation. We assume that the TLR polymorphism effect on the female reproduction traits can be mediated by non-immune functions of TLRs in myometrial signalling rather than indirectly by changes in the infection resistance, consistently with the known effects of TLRs in model species. However, prevailing associations with non-coding or synonymous SNPs point at the location of causal variation in the regulatory regions of the TLR genes.