Northern Poland is inhabited by native Apis mellifera mellifera (AMM) and the non-native A. m. carnica (AMC) which was introduced by beekeepers. However, hybrids between the two subspecies of honey bee are relatively rare. The lower than expected proportion of hybrids is hypothesised to be related to reproductive isolation between AMM and AMC. To verify this hypothesis, we allowed the AMM and AMC queens to be naturally inseminated in an area inhabited by both AMM and AMC drones. Genotype of the queens and their sexual partners were derived based on random samples of their worker offspring. Assignment of parental genotypes to the two subspecies was performed with a Bayesian clustering method. In colonies headed by AMM queens, workers were fathered mainly by AMM drones. On the other hand, in colonies headed by AMC queens workers were fathered by drones of both subspecies. The partial reproductive isolation reported here between AMM and AMC may facilitate conservation of the declining population of AMM.