The two-spot ladybird Adalia bipunctata (Linnaeus, 1758) (Coleoptera: Coccinellidae) is a widely distributed generalist, feeding preferably on aphids. The species inhabits various environments, including anthropogenic landscape, where pollution and microclimate enables to attain very high numbers of favorite prey. Recently, the abundance of the two-spot ladybird has declined, mainly as an after-effect of invasion of the competing species — the harlequin ladybird Harmonia axyridis (Pallas, 1773). High phenotypic polymorphisms made the two-spot ladybird classic model for population genetic studies of Coccinellidea. However, studies investigating diversity of the species at DNA level have Coccinellidae been scarce. In our investigation we used microsatellite markers to characterize genetic diversity in the two-spot ladybird populations from different environments. We made an attempt to amplify 13 loci, identified previously in the genome of the species, however due to lack of amplification, monomorphisms and presence of the null alleles, the final set of six markers useful for analysis on a population level was obtained. Analyzing genotypes of 124 individuals, we confirmed high genetic diversity of the species, suggested previously by studies, applying allozymes and mitochondrial DNA. Also, microsatellites revealed weakly pronounced genetic structure in a large scale (localities separated by a distance of 500 kilometers). However, we found some indications (signs of the Wahlund effect) that gene flow could be disturbed among sites in diverse environments (rural, suburban and urban). Our data suggested that invasion of the harlequin ladybird has not yet affected populations of the two-spot in such extent to alter genetic diversity or genetic structure.