A honey bee queen mates with a number of drones a few days after she emerges as an adult. Spermatozoa of different drones are stored in her spermatheca and used for the rest of the queen’s life to fertilize eggs. Sperm usage is thought to be random, so that the patriline distribution within a honey bee colony would remain constant over time. In this study we assigned the progeny of a naturally mated honey bee queen to patrilines using microsatellite markers at the queen’s age of two, three and four years. No significant changes in patriline distribution occurred within each of two foraging seasons, with samples taken one and five months apart, respectively. Overall and pair-wise comparisons between the three analyzed years reached significant levels. Over the three-year period we found a trend for patrilines to become more equally represented with time. It is important to note that this study was performed with a single queen, and thus individual and population variation in sperm usage patterns must be assessed. We discuss long-term changes in patriline composition due to mixing processes in the queen’s spermatheca, following incomplete mixing of different drones’ sperm after mating.