The genus Proteus currently only encompasses two subspecies: P. a. anguinus and P. a. parkelj. It has been known for more than a quarter of a century that this division of the species on the basis of morphological signs is no longer good enough. On the basis of alloenzymes, mitochondrial DNA, core DNA and also a more detailed overview of external morphology, genetic differences between isolated populations have been demonstrated. In this work we take a more detailed look at the southwestern region of Slovenia, where only the white subspecies of P. a. anguinus is present. Current research has evaluated the southwest population as a deeply separated from others, with evidence for further differentiation within the population. The main reason for the diverse genetic structure in the area could be attributed to the geographic barrier represented by two different drainage basins: the Adriatic and Danube. In order to determine the affiliation to the population of individual samples, the mitochondrial DNA was used and short tandem repeats of nuclear DNA. The main population genetic analysis was carried out using the Bayesian approach using the MCMC algorithm. Affiliation to individual populations was also assessed by genotyping with restriction analysis of the control region of mitochondrial DNA. The results thus showed that in the southwestern region of Slovenia in the vicinity of Sežana, due to the complicated underground flows and rare hydrological events in the Reka river two distinct lines of proteus come into contact: Karst PSV and the eastern karst Ljubljanica population. Between these two populations there is a contact zone where hybridization occurs in a narrow area in the cave Jama Sežana River.