The Internal Transcribed Spacer (ITS) region of the nuclear ribosomal repeat was investigated in the search for a suitable genetic marker for population studies of the noble crayfish Astacus astacus (L.). DNA sequencing revealed the presence of two microsatellite insertions in ITS1. By designing highly specific PCR primers, adjacent to one of the insertions, we were able to use fragment analysis to explore the variation of the insertion in 642 specimens from 17 populations of crayfish from Sweden and former Yugoslavia. A new statistical test, the Population Divergence Test, was developed to assess statistical significance of divergence between populations. This test does not assume Mendelian inheritance. Our results demonstrate that different populations often produce characteristic fragment patterns, and that most, but not all, populations are genetically distinct, with high significance. The populations that cannot be significantly differentiated are situated in close geographic proximity to each other and belong to the same main river system, probably reflecting that these populations have had recent contact and that gene flow has occurred.