Balkan loach (Cobitis elongata Heckel & Kner, 1858) is freshwater fish from the Cobitidae family which is endem of Danube watershed and protection of their populations is necessary. Phylogenetic relationship, the possible connection between population through migrations, as well as the evolution of this species are not sufficiently researched and there is not enough data about whole areal of prevalence. The aim of this study was to: establish the phylogenetic and phylogeographic relations of the balkan loach to identify intraspecific structure, to determine genetic diversity of certain populations and the whole species, estimate the effective size of the individual populations and the whole species, clarify the evolution history and determine the approximate period of occurrence of this species as well as the divergence of individual populations. In order to determine intraspecific structure of balkan loach, a phyolgenetic reconstruction was conducted based on the mitochondrial gene sequence for cytochrome b using the several methods: maximum parsimony, Bayesian method and median joining. Using the DnaSP computer program, the measures of the genetic polymorphism are determined and with MIGRATE migration rate among the population. Divergence times are determined using Beast software. The results obtained by these methods have shown that the species is deeply structured and that there are four groups of haplotypes. Also, low intraspecific and intrapopulation diversity was determined along with genetic isolation of population and low flow of genes. Small effective population sizes and poor population connectivity are the reflection of ecology of this species.