At the present ecological surveys and ecological monitoring require accurate and rapid identification of biodiversity level without using traditional morphology analysis. To solve this problem a range of molecular ecological methods was developed, particularly the DNA-barcoding technique. However the detection of barcode gap might be challenging due to false-positive and false-negative errors. In this paper, a set of bioinformatic methods providing objective estimation of difference between intraspecific and interspecific variance was tested. The set of nucleotide sequences of the mitochondrial gene fragment for cytochrome c oxidase I (COI) of morphologically indistinguishable Siberian was used as a model object. The GMYC application showed intraspecific variance threshold at 1.85 %. Using ABGD algorithm allowed to detect a reliable barcode gap of at least 4 %. Both methods independently divided the analyzed set of sequences into 10 phylogenetic groups, where 68 sequences of Siberian leeches formed a separate cluster. The lack of morphological and genetic similarity to previously described species may indicate the discovery of a new species of annelid worms. At the same time it was shown that the sequences previously attributed to species Erpobdella ochoterenai actually belong to 4 different species which suggest the necessity of recurrent morphological analysis of this group. Additionally, both GMYC and ABGD unambiguously assigned previously unidentified sample from Macedonia to species E. vilnensis, which demonstrates the utility of the DNA-barcoding concept. This allows to consider bioinformatic approach to molecular ecology as a suitable tool for objective species delimitation particularly within Erpobdellidae, significantly simplifying the determining of annelid species relationships, which will enhance ecological surveys, increase the efficiency of monitoring and our knowledge for using biodiversity.