The negative effects of habitat fragmentation and population isolation on population viability, genetic variability and structuring are well documented, and conservation plans failing to take into account spatial population structure and connectivity can be ineffectual. Of special concern are populations at the periphery of the species range that might show reduced genetic diversity, thus affecting their adaptive potential at environmental margins. We investigated genetic variability and differentiation of the globally near threatened and locally endangered fire salamander Salamandra infraimmaculata in northern Israel, an area that represents the periphery of this species' distribution range. Analyses of variability in 15 microsatellite loci from 20 sites revealed substantial population structuring, most of which was due to a strong subdivision between two regions separated by a heavily urbanized valley. In addition, levels of genetic variability within populations were lowest in the peripheral, southernmost populations. These results suggest that the conservation plans for this species should recognize the lower diversity and increased divergence in the peripheral regions, and take into account the observed spatial population structure when devising strategies and measures to ensure the species persistence.