In the grasshopper Podisma pedestris, units of the ribosomal DNA (rDNA) multigene family are not identical, but comprise multiple genetic variants. We surveyed this variation using a novel pyrosequencing approach. The history of the study populations is well characterized as the pattern of colonization can be inferred from the distribution of two chromosomal races that invaded from different directions after the last glacial maximum and finally met to form a hybrid zone. This knowledge of the populations' ancestry allows us to draw inferences about the rate of change in rDNA composition. The rDNA data have, in turn, been revealing about the populations' ancestry, indicating a previously unsuspected route of postglacial colonization. The two chromosomal races were found to have genetically distinctive rDNA composition, demonstrating the persistence of differences for thousands of generations. It follows that the hybrid zone represents a natural experiment in which repeated crossing and backcrossing between these different rDNA lineages has occurred for over 8000 generations. The association between chromosomal race and rDNA composition has been broken down within the zone. It therefore appears that rDNA variants move freely across the zone and are not under opposing selection pressures in the two races, as had previously been suspected.