Wood ant colonies that appear to consist of individuals representing different species are described in several previous papers. The present study is the first to elucidate the genetic basis of the spectacular morphological variability observed within such colonies. Two seemingly mixed colonies (FM-1 and FM-2) from southern Finland were investigated. On the basis of the morphology of their workers these colonies were comprised of individuals with phenotypes typical of Formica rufa L., F. polyctena Först., and F. aquilonia Yarr. The sequence of an mtDNA fragment (5' end of the cytochrome b gene) was used to examine the phylogenetic relationships among haplotypes of workers from homogeneous colonies of different wood ant species and the two supposedly mixed colonies, and to sort the individuals within the colonies into matrilines. Six microsatellite loci were used to analyse the genetic differentiation between colonies and among workers within colonies, and to detect putative hybrids. The results show that, independently of their phenotype, workers from the "mixed" colonies were genetically more similar to other individuals in their colony than to those in the homogeneous F. rufa, F. polyctena or F. aquilonia colonies. However, while colony FM-1 consisted of offspring of the same queen or more likely several maternally related queens, colony FM-2 consisted of the offspring of at least four unrelated queens. The data suggest hybridisation between F. polyctena × F. aquilonia and F. polyctena × F. rufa (and possibly subsequent mating between these hybrids) as the most probable mechanism leading to the existence of these two colonies, which implies that the hybrids are fertile. This study shows that colonies of wood ant hybrids can arise spontaneously and persist under natural conditions. The results also revealed that even some morphologically homogeneous colonies are genetically heterogeneous. In the case of closely related, morphologically similar species that interbreed, morphology can be a bad predictor of genetic differences between individuals.