Cytochrome P450 3A (CYP3A) subfamily enzymes are involved in the metabolism of about 50% of drugs in clinical use. The most important are CYP3A4 and CYP3A5 enzymes, which are characterised by interindividual variability. Genetic variants can explain part of the variability of CYP3A activity. The clinically most important are variants CYP3A5*3 and CYP3A4*22, while the CYP3A4*1B is less important. Data on the frequency of CYP3A polymorphisms in the Croatian population exist, but they are not from the same dataset. In this study, the frequencies of CYP3A4*1B, CYP3A4*22 and CYP3A5*3 were investigated in the same data set in the Croatian population. DNA samples were extracted from whole blood samples over eight years of routine pharmacogenetic testing at the University Hospital Centre Zagreb. A total of 1100 subjects were included in this study. Genotyping of CYP3A4*1B, CYP3A4*22 and CYP3A5*3 was performed by the TaqMan® real-time PCR method. CYP3A allele frequencies were CYP3A4*1B=0.0698 (n=258), CYP3A4*22=0.0355 (n=1100) and CYP3A5*3=0.9645 (n=440). The analysis of genotypes showed: (i) for CYP3A4*1B, 86.43% of subjects were carriers of genotype *1/*1, 13.18% of genotype *1/*1B, and 0.39% of genotype *1B/*1B; (ii) for CYP3A4*22, 93.09% are carriers of the *1/*1 genotype, 6.73% of the *1/*22 genotype, and 0.18% of the *22/*22 genotype; (iii) for CYP3A5*3, 83.18% of respondents are carriers of the *3/*3 genotype, 16.14% of the *1/*3 genotype, and 0.68% of the *1/*1 genotype. In this study, the frequencies of all investigated polymorphisms were slightly higher than in previous studies conducted on the Croatian population. The frequency of the CYP3A4*1B, CYP3A4*22 and CYP3A5*3 variant alleles and genotypes in the Croatian population is in accordance with the other European populations.