Geographic variation within species can originate through selection and drift in situ (primary variation) or from vicariant episodes (secondary variation). Most patterns of subspecific variation within European flora and fauna are thought to have secondary origins, reflecting isolation in refugia during Quaternary ice ages. The bushcricket Ephippiger ephippiger has an unusual pattern of geographical variability in morphology, behaviour and allozymes in southern France, which has been interpreted as reflecting recent primary origins rather than historical isolation. Re-analysis of this variation using Geographical Information Systems (GIS) suggests a possible zone of hybridization within a complex pattern of geographical variation. Here we produce a genetic distance matrix from restriction fragment length polymorphism (RFLP) bandsharing of an approximately 4.5 kb fragment of mitochondrial DNA (mtDNA), and compare this with predictions resulting from the GIS analysis. The mtDNA variation supports a postglacial origin of geographical variation. Partial Mantel test comparisons of genetic distances with matrices of geographical distance, relevant environmental characteristics and possible refugia show refugia to be the best predictors of genetic distance. There is no evidence to support isolation by distance. However, environmental contrasts do explain significant variation in genetic distance after allowing for the effect of refugial origin. Also, a neighbour-joining tree has a major division separating eastern and western forms. We conclude that the major source of variation within the species is historical isolation in glacial refugia, but that dispersal, hybridization and selection associated with environmental features has influenced patterns of mtDNA introgression. At least two valid subspecies can be defined.