The human UDP-glucuronosyltransferase 1A9 (UGT1A9) plays a central role in the metabolism of different therapeutic drugs, carcinogens and endobiotics. The UGT1A9 gene shows genetic polymorphism with frequencies significantly different in populations and ethnic groups. Many of these genetic variants are directly responsible for polymorphic drug metabolism. Three crucial alleles of UGT1A9, UGT1A9*3 (p.Met33Thr), *4 (p.Tyr242X), *5 (p.Asp256Asn) are associated with decrease or absence of enzyme activity, which intensify the risk of toxic effect during biotransformation. The goal of the present study was to discover frequencies of these genetic variations in 308 healthy individuals representing Polish population. The genotypes were determined by pyrosequencing. We demonstrated that the frequency of the variant UGT1A9*3 was 0.016, which suggests the need for detailed analysis of its effect on important drugs metabolism level in Polish population. Alleles UGT1A9*4 and UGT1A9*5 were not present in any of the subjects. So far, no studies have been conducted in which the distribution of these alleles has been determined in the Polish population.