Urbanised areas are capable of exerting a strong impact on the distribution of genetic diversity within populations of animals. Urban invertebrate species are currently either relicts from pre-urban ecosystems, or have immigrated during or following urbanisation. We analysed 10 microsatellite loci in 196 specimens of the harlequin ladybird (Harmonia axyridis), an invasive species in Poland sampled in three of the country's cities. Of Asiatic origin, this ladybird has been spreading through other continents, including Europe. Results showed that the Polish cities are being invaded by harlequin ladybirds that are uniform in terms of their genetic pool, with no significant genetic differentiation present between the urban populations investigated. Rapid spread and what are probably the large numbers of individuals colonising new areas allow this species to maintain high genetic diversity and avoid bottleneck effects. However, we suggest that urban populations differ in terms of genetic diversity. The highest genetic diversity characterises the most recently invaded area of Olsztyn in the north of Poland. Genetic data further suggest that this population could be still in an expansion phase. The earliest colonising population in Wrocław exhibits signs of a Wahlund effect, suggesting that gene flow among local groups within this urban area could be disturbed or impaired. We conclude that, in the case of an urban population of the harlequin ladybird, successful colonisation is followed by the onset of a decline in genetic diversity, with isolation between local sampling sites appearing. Further studies are required, however, if this process is to be elucidated.