The solution covers reaction for the molecular diagnostics of the variants of TLR8 in milking cattle. The importance of the TLR8 gene is determined by its participation in the early immunity against the viral diseases of cattle. The area of the technology can be generally denoted as veterinary diagnostics. The design of the set of reactions is derived from the general technique of primer extension which exploits the first step in the enzymatic synthesis of the complementary strand of DNA for the determination of the type of nucleotide. The framework for the design of the procedure was the bioinformatic and population-wide estimate of the variability in the immunity genes of the TLR family in the milking cattle population. The second part of the protocol development included the design of the genotyping primers and the optimization of the genotyping reactions. The integral panel of reactions targets those genetic polymorphisms that are spread in the Central European population of milking cattle, achieving in this way the reduction in costs. The set eliminates the disadvantages of the former methods for the polymorphism screening in this group of TLR and adds a value of the optimization for the given population. The selection of SNP follows the distribution of the polymorphism in the representative breeds of the Czech Red and the Czech Red Pied cattle. The part of the developed solution is the optimization of the conditions for parallel realization of the multiplexed genotyping reactions in the groups of 5 and 6, which markedly decreases the labour and financial costs of the method.