Genetic differentiation of Rana temporaria from the Pyrenean and Cantabrian mountains in Spain was studied by means of allozyme electrophoresis. 24 loci were analysed in 104 specimens from 15 populations: nine populations from the Pyrenean massif, five populations from the area of the Cantabrian mountain chain (regions of Galicia, Asturias, and Basque Country), and one population from Germany. Three distinct clusters were distinguished by phenetic analysis: (a) the Pyrenean samples and the single population from the Basque Country, (b) the populations from Galicia and Asturias) and (c) the German population. Ordination (PCA) resulted in one principle component (PC1) that separated Cantabrian from Pyrenean populations, and in a second one (PC2) that separated the single German population from the Iberian ones. PC1 indicated introgression that was corroborated by west-east clines in several alleles along the Cantabrian chain. The rather clear separation of the Cantabrian and Pyrenean clusters (mean genetic distance 0.121) suggests that two genetically different subspecies of R. temporaria may be distinguished in Spain. The absence of fixed allelic differences between populations refutes recent hypotheses of the existence of syntopic sibling species within R. temporaria in Spain. Biogeographically, the Pyrenean and Cantabrian populations possibly originated in two separate colonisation events starting from different glacial refuges. The strong morphological differentiation of Pyrenean R. temporaria populations is not paralleled by genetic divergence, and may better be explained by ecological factors such as climate, altitude and vegetation.