The natural reproductive behaviour of invasive insects is pivotal knowledge for managing species of ecological or economic concern. We use microsatellites to examine female multiple mating and multiple nest infestations in the introduced parasitic fly, Philornis downsi, which causes high mortality in endemic birds on the Galápagos Islands. We analyse larvae and pupae within 57 nests from Santa Cruz and Floreana Islands in both the highland and lowland habitats. Sib-ship reconstructions of offspring revealed that up to five females may infest a single nest, while multiple mating in females was frequent (65% of reconstructed maternal genotypes), with an average of 1.91 (±0.06 SE) males per female. Genetic relatedness (R) of offspring within nests was generally low, though lowland nests on Floreana had higher R than highland nests. Knowledge of the reproductive behaviour of P. downsi is necessary for modelling appropriate management strategies, in particular, the sterile insect technique, for which success is greatly influenced by female multiple mating.