Nuclear genetic diversity and differentiation of 341 sheep belonging to 12 sheep breeds from Croatia and Bosnia and Herzegovina were examined. The aim of the study was to provide the understanding of the genetic structure and variability of the analysed pramenka sheep populations, and to give indications for conservation strategies based on the population diversity and structure information. The genetic variation of the sheep populations, examined at the nuclear level using 27 microsatellite loci, revealed considerable levels of genetic diversity, similar to the diversity found in other European indigenous low-production sheep breeds. Population-specific alleles were detected at most loci and in breeds analysed. The observed heterozygosity ranged from 0.643 (in Lika pramenka) to 0.743 (in Vlasic pramenka), and the expected heterozygosity ranged from 0.646 (in Lika pramenka) to 0.756 (in Dalmatian pramenka). Significant inbreeding coefficients were found for half of the populations studied and ranged from 0.040 (Pag island sheep) to 0.091 (Kupres pramenka). Moderate genetic differentiation was found between the studied sheep populations. The total genetic variability observed between different populations was 5.29%, whereas 94.71% of the variation was found within populations. Cres island sheep, Lika pramenka and Istrian sheep were identified as the most distinct populations, which was confirmed by the factorial analysis of correspondence and supported through a bootstrapping adjustment to correct for the difference in the sample sizes. The population structure analysis distinguished 12 clusters for the 12 sheep breeds analysed. However, the cluster differentiation was low for Dalmatian, Vlasic, Stolac and Krk pramenka. This systematic study identified Lika pramenka and Rab island sheep as those with the lowest diversity, whereas Istrian sheep and Pag island sheep had the highest. Conservation actions are proposed for Istrian, Rab and Cres island sheep, Lika and Kupres pramenka because of high estimated coefficients of inbreeding.