The molecular DNA variation among Salamandra infraimmaculata larvae populations, representing eight breeding sites in Israel, was studied. Samples from larvae were analyzed by sequence analysis of the mitochondrial cytochrome b fragment and D-loop regions (GenBank accession nos. EU852723–EU852738). A neighbor-joining analysis had an optimal arrangement with a branch length sum of 0.00652338. The genetic distances, which were computed by the maximum composite likelihood method and which are presented as the number of base substitutions per site, demonstrated that there are two sub-populations. One consists of larvae from unpredictable breeding sites, of which most are winter ponds, and the other one includes populations from perennial water sources, mostly streams and springs.