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Restriction fragment length polymorphism (RFLPs) of the mouse period-homologous sequence were studied in 13 populations of the four chromosomal species (2 n = 52, 54, 58 and 60) of the mole rat, Spalax ehrenbergi superspecies in Israel. The period locus of Drosophila melanogaster is implicated in controlling the circadian rhythm as well as the male courtship song rhythm. Multiple DNA homologies exist in the mole rat and correspond to more than 10 loci. The level of polymorphism is very high, with a large number of alleles per locus, increasing from the northern to the southern species along a gradient of increasing aridity. Variation was also found in an isolated desert population, with a unique fragment specific to this population. Fragment variation allows distinction between chromosomal species, and confirms earlier evidence that gene flow does not occur between them. A correlation was found between some allelic fragments and the number of apparent harmonics of the courtship calls. This finding suggests an interesting testable hypothesis that the existence of a locus (homology) is responsible for the courtship call parameters.