We investigated the mating and fertilization patterns in one Czech local population of the invasive aphidophagous ladybird Harmonia axyridis (Pallas), sampling the same mixed generations before and after overwintering. Our study using eight microsatellite loci provides the first direct evidence that females of H. axyridis are highly polyandrous in the wild and that they not only mate multiple times but also use the sperm from multiple males to fertilize their eggs. Three types of values (minimum number of fathers, estimated number of fathers and effective number of fathers contributing to progeny of one mother) were about two times higher in the spring sample (2.9, 5.2, 3.1), in the peak of their reproduction period, than in the preceding autumn sample (1.6, 2.8, 1.6). The contribution of individual fathers to the number of progeny was highly unbalanced, with a few dominating fathers and many fathers with low number of progeny.