Croatia is diverse with earthworm species and the Croatian territory, as part of Balkan Peninsula, is considered to be one of the divergency centers of genera Octodrilus and Octodriloides. So far, 15 species of the genera Octodrilus and 5 species of the genera Octodriloides have been recorded in Croatia. The research was conducted in order to determine the genetic and species diversity of the mentioned generas by DNA barcoding 177 samples from 77 localities throughout Croatia. The COI region from mtDNA was isolated, amplified and sequenced from the sample’s tissue. Using the SEQUENCHER (4.1.4) and MEGA-X computer programs, the sequences were aligned and processed and a phylogenetic tree was constructed according to the maximum-likelihood method. A total number of 28 lineages were singled out from a tree and classified to 9 species of the genera Octodrilus and 3 species of the genera Octodriloides. 3 lineages of the genera Octodrilus were not classified to described species due to specificity in structure or juvenile stage of samples. 5 species form a complex of at least two lineages with significant genetic distances. The research also showed the advantage of determining earthworm species by molecular methods instead of determination according to morphology and anatomy, which is very complex. It is necessary to conduct researches on a far larger number of samples in order to detect the remaining described species in Croatia by molecular-phylogenetic methods and to determine their phylogenetic position by comparing them with the results of this research.