This study evaluates the utility of DNA barcoding (mitochondrial cytochrome oxidase subunit I; COI) as a biodiversity and conservation applied tool for identifying fish fauna from the southeastern Mediterranean (the continental coast of Israel), a hot-spot area for biological invasion, also with an eye to establish the foundation for follow-up studies that will use environmental DNA (eDNA) tracks of native and invasive species, and for the application of eDNA concepts and methodologies in nature conservation. We established a dataset of 280 DNA barcodes, representing 110 marine fish species (all identified by a taxonomist), belonging to 75 native and 35 Lessepsian migratory species that were tested within and against the BOLD system database. Most of the query sequences showed 98% matches with reference DNA barcodes in the BOLD system excluding two Trachurus species, three Dasyatis pastinaca and two Rhinobatos rhino batos individuals. Relatively high intraspecific genetic distances were observed in two Elasmobranchii species (8.83%-18%), suggesting possible cryptic species. In contrast, relatively low interspecific genetic distances were found between two Actinopterygii species (1.54%). Gobiidae family members were observed as being the most scattered on the Bayesian tree. Out of the 110 fish species, 48 were taxonomically discordant with the BOLD BINs (4 at the family level, 15 at the genus level and 29 at the species level), 53 were concordant and 9 were singletons. Discordancy was noted for some Dip lodus and Fistularia species during the BOLD BIN analysis. Apogon queketti, Champsodon nudivittis and Cheilodipterus novemstriatus were uploaded to the BOLD system for the first time. We elucidated 177 haplotypes, 122 for the native fish species and 55 for the Lessepsian species. The results will allow the evaluation of invasive species success and will help developing improved policies for the conservation of Mediterranean biodiversity. (C) 2017 Elsevier GmbH. All rights reserved.