The main objective of this study was to investigate the genetic polymorphism of the interleukin-1-receptor associated kinases 1 and 4 (IRAK1, IRAK4) and the myelin and lymphocyte protein gene (MAL) in the Czech Pied cattle (CP) population. Very little is known about the significance of these genes in cattle species, especially for infectious resistance, which may be crucial for breeding. Genetic variability was determined by next-generation sequencing (NGS) by a screening of 164 CP bulls. The obtained results of PacBio sequencing technology (Pac Bio RSII, Pacific Biosciences) were recorded and compared with database polymorphism. Seventeen single nucleotide variations (SNV) were found in the IRAK1 gene, three deletions and 12 SNVs were found in the IRAK4 gene. The MAL gene contained only two SNPs. Bioinformatics prediction of the functional impact of polymorphism found in the population identified only one non-synonymous 5131 A/G mutation in the IRAK1 gene, which leads to amino acid substitution. To determine population variability three genotyping methods were used: the Sanger sequencing method, the DNA microarray method (Illumina), and the primer extension method (SNaPShot, Thermo Fisher Scientific)., Comparison of the SNP rs110533802 allelic frequency between the CP and other commercial cattle breeds was performed in cooperation with the Czech-Moravian Society of Breeders. Based on the obtained data, the functionally significant variability of the examined genes can be considered rather limited in the investigated population of the CP.