Rana iberica is an Iberian endemic with populations mainly distributed in northwestern Iberia, but with scattered populations along the Sistema Central in central Spain. These latter populations inhabit mostly mountain areas and are endangered due to habitat loss and fragmentation. At present, no information about genetic variability and gene flow patterns between populations is available and, consequently, it is unknown to what extent geographical fragmentation might have caused restrictions to gene flow. We used six microsatellite loci to characterise genetic variability in R. iberica populations from the Sistema Central. We analysed samples of 142 specimens from 15 populations in this region and another two from additional locations in the Iberian Peninsula. The number of alleles per locus observed ranged from four to 15. Allelic richness, percentage of private alleles and gene diversity was significantly lower in populations from the Sistema Central, where inbreeding coefficients were relatively high. Analysis of molecular variance (AMOVA) and estimated FST values indicated the existence of genetic substructuring within this region and suggested the existence of restrictions to gene flow in geographically close populations. Low heterozygosities and a trend towards lower values of genetic variability from western to eastern populations within the Sistema Central suggest that R. iberica has colonised this region recently. We hypothesise that the observed pattern is due to the interaction of both historical (reduced genetic variability in peripheral populations) and present-day (anthropogenic habitat loss) factors that confirm the vulnerable status of populations of R. iberica in central Spain and point to the need for adopting measures directed towards the preservation of existing habitat.