The aim of this study was to observe sperm utilization in Apis mellifera ligustica naturally mated honeybee queens over a two year period. The study was conducted by microsatellite analysis of the progenies of 6 honeybee queens sampled during two consecutive egg-laying seasons (from June to October in the first year and from March to June in the second year). Four microsatellite markers were initially analysed. However, the most variable locus A76 alone was sufficient to distinguish the genotype of the queens and the patrilines of the six colonies. The effective number of matings based on the frequencies of the patrilines and corrected for the sample size, was computed and was 8.10, slightly lower than the number found by other authors. No significant differences were noted in the observed frequencies of the patrilines in the two reproductive seasons. Moreover, there was a similar distribution of the patrilines in the first month (June 2002) with respect to the last month (June 2003). These results indicated that there was a temporal conservation of the genetic structure of the colonies.