We provide a comparative population genetic study of the elusive amphibian, Proteus anguinus, by comparing the genetic diversity and divergence among four cave populations (96 individuals) sampled in the Dinaric Karst of Croatia. We developed 10 variable microsatellite markers using pyrosequencing and applied them to the four selected populations belonging to four different cave systems. The results showed strong genetic differentiation between the four caves corroborating with previous findings suggesting that Proteus might comprises several unrecognized taxa. Our results confirmed that gene flow should be high within the caves, whereas it is low between hydrographic systems since geological periods. Finally, we conclude that the high genetic subdivision suggests the necessity of treating the four studied Proteus populations as evolutionary significant units.