Differentiation among the European populations of the six-toothed spruce bark beetle Pityogenes chalcographus was observed for the first time in the 1970s as mating males from Northern Europe with females from Central Europe led to a significant decline in fecundity. Morphological examination revealed that P. chalcographus can be separated into two European races. Here, we investigated the genetic background of this separation by analysing 39 populations (n = 695), sequencing almost the complete cytochrome oxidase I gene (1543 bp) and applying single-strand conformation polymorphism. Phylogenetic analysis of 58 haplotypes yielded three major clades with a maximum sequence divergence of 2.33%, indicating that the demographic events took place in the late Pleistocene. These results support the hypothesis of allopatric divergence of the mtDNA lineages, which postglacially came into sympatric existence in Europe. However, as a result of partial crossing incompatibility the diverged lineages retained their genetic identity during postglacial times. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 331–340.