The number of previous anthropological studies pointed to very complex ethnohistorical processes that shaped the current genetic structure of Croatian island isolates. The scope of this study was limited to the general insight into their founding populations and the overall level of genetic diversity based on the study mtDNA variation. A total of 444 randomly chosen adult individuals from 32 rural communities of the islands of Krk, Brač, Hvar and Korčula were sampled. MtDNA HVS-I region together with RFLP sites diagnostic for main Eurasian and African mtDNA haplogroups were analysed in order to determine the haplogroup structure. The most frequent haplogroups were »H« (27.8–60.2%), »U« (10.2–24.1%), »J« (6.1–9.0%) and »T« (5.1–13.9%), which is similar to the other European and Near Eastern populations. The genetic drift could have been important aspect in history, as there were examples of excess frequencies of certain haplogroups (11.3% of »I« and 7.5% of »W« in Krk, 10.5% of »HV« in Brač, 13.9% of »J« in Hvar and 60.2% of »H« in Korčula). As the settlements on the islands were formed trough several immigratory episodes of genetically distant populations, this analysis (performed at the level of entire islands) showed greater genetic diversity (0.940–0.972) than expected at the level of particular settlements