Most neuropteran species have similar male genitalia which makes their morphological identification difficult. The aim of this study was to investigate potency of the cytochrome c oxidase subunit I (COI) gene-based marker to distinguish twenty-one morphospecies members of Neuroptera and to reveal their phylogenetic relationships. The samples representing four neuropteran families (Ascalaphidae, Myrmeleontidae, Nemopteridae and Chrysopidae) were collected from geographically separated cities (Kamyaran, Saghez, Mari van, Sanandaj, Divandareh and Baneh) from the west of Iran. Initially, the samples were identified morphologically and then subjected to DNA extraction, PCR assay and DNA sequencing. Accordingly, seven morphological species including Chrysopa pallens, Suarius nanus, Distoleon nuristanus, Acanthaclisis occitanica, Iranoidricerus iranensis, Nemoptera rachelli and Halter nutans were identified. Analysis of genetic similarity among Neuropterans showed that Suarius nanus and Chrysopa pallens species from Chrysopidae had the most dissimilarities with the other species. Results of phylogenetic analysis corroborated these finding in which S. nanus and C. pallens formed a monophyletic clade and were separated from the other species. Interestingly, Nemoptera rachelli and Halter nutans from Nemopteridae separated genetically from each other which shows their different genetic pattern. Analysis of nucleotide conversion rate for COI sequences showed that the most and the least conversion rates occurred between T-A and C-G bases, respectively. Pairwise genetic analysis showed the most inter-species variation in S. nanus and C. pallens. Accordingly, Distoleon nuristanus, Acanthaclisis occitanica, and Iranoidricerus iranensis showed the most intra-species variations. As a conclusion, the COI gene-based marker could produce a clear-cut resolution among these morphospecies insects similar to what should be obtained from their morphological classification.