The main objective of this study was to investigate gene polymorphism of interleukin 1 receptor-associated kinase 1 and 4 (IRAK1 and IRAK4) in Czech Pied Cattle (CP). There is a minimal exploration of the IRAK1 and IRAK4 genes especially concerning infectious resistance, which can be essential for the cattle breeding program. The genetic variability of IRAK1 and IRAK4 was determined utilizing of next-generation sequencing (NGS) of 164 bulls representing the breed’s gene pool. The mixture of amplicons based on 25 pairs of primers covering whole sequences of these genes was resequenced with PacBio technology (Pac Bio RSII, Pacific Biosciences). Obtained NGS results were assigned to the bovine sequence UMD_3.1.1 and compared with a public variation database (EBI) content. Seventeen single nucleotide polymorphisms (SNPs) were found in IRAK1, and three deletions and 12 SNPs were found in the IRAK4 pool.