The family Gobiidae is the largest teleost family, consisting of more than 2,000 species in more than 200 genera. They have adapted to a wide range of habitats, inhabiting mostly marine and brackish waters. Recently invasive Ponto-Caspian gobies have caused significant changes to the structure of fish assemblage throughout the European waters. The aim of the study was to verify the possibility of identifying three species of the genus Gobiidae based on the analysis of the mitochondrial region of the cytochrome oxidase subunit 1 (COI) gene. 339 samples originated from seven countries and 17 different sites were investigated. Qualitative and quantitative assessment of the extracted DNA was conducted by measuring light absorbance and electrophoretic separation. Approximately 650-bp (base pairs) fragments were amplified from the 5' region of the mitochondrial COI with the PCR (polymerase chain reaction) reaction. Sequences of 652 bp were obtained and compared with reference sequences in the National Center for Biotechnology Information (NCBI). Based on the sequence analysis, we determined that in the analysed geographical regions, the goby preliminary identified morphologically as the round goby was represented by three species: round goby ( Neogobius melanostomus), tubenose goby ( Proterorhinus marmoratus), and monkey goby ( Neogobius fluviatilis). The latter two are newly reported for their new habitats. Genetic characterisation of such populations offers the opportunity to assess their genetic stability, which provides agencies managing the aquatic environment in EU countries with a tool for revising the legislation, including regulations relevant for updating the status of native and invasive species in European waters.