Internal Transcribed Spacer (ITS) is one of the most used barcoding regions for the molecular phylogenetics and barcoding of orchids. Our aim in this study is to test the reliability of ITS on barcoding of closely related Neotinea spp., including Neotinea tridentata, Neotinea ustulata subsp. ustulata and Neotinea ustulata subsp. aestivalis, by comparing it to the accD-psaI intergenic spacer of the plastid DNA. Both ITS and accD-psal regions were amplified by specific primer sets and sequenced. Phylogenetic trees were regenerated by using Maximum Parsimony approach. The results showed that ITS separated some A'. tridentata samples of Turkish, Greek, Hungarian and Croatian samples from the others on the phylogenetic trees due to the incomplete lineage sorting. In contrast to ITS, the accD-psal marker could successfully separate :V. tridentata and N. ustulata samples according to a priori species classification. Our findings refer to a hybridisation story between some N. tridentata and N. ustulata. We propose not to use ITS sequences directly as a barcode and to reconstruct the phylogeny of the Neotinea group. Instead, the inclusion of other nuclear regions such as LFY, ADH etc., or utilisation of whole genome sequencing could give better barcoding results.