CYP2B6 is one of the numerous members of the cytochrome P450 family and constitutes 2% to 10% of the total hepatic cytochrome content in humans. It is responsible for the metabolism of 4% of the top 200 drugs and is inducible by drugs and other xenobiotics. CYP2B6 gene expression is highly variable among individuals and significant differences among populations have been detected. In this study, the presence of mutations at seven CYP2B6 loci (rs12721655, rs2279343, rs28399499, rs34097093, rs3745274, rs7260329 and rs8192709) was analysed in three Croatian Roma populations from the regions of Baranja, Međimurje, and Zagreb. The KASP (Kompetitive allele specific PCR) method was used in loci genotyping. Mutated alleles were detected at loci rs2279343, rs3745274, rs7260329 and rs8192709. Mutation frequencies were different among analysed populations and significant differences in genotype frequencies among populations were detected. All analysed polymorphous loci in all populations are in Hardy-Weinberg equilibrium. Significant linkage disequilibrium in pairs of loci in all three analysed populations has been documented. This study confirms unique genetic features of Roma populations, which in this specific case could affect pharmacotherapy modulation.