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Despite being one of the most speciose fish families in the Mediterranean Sea, knowledge about the diversity of gobies (Actinopterygii: Gobiidae) in this sea is still unsatisfactory, as documented by recent descriptions of a number of new species. Although very common in shallow water, Gobius incognitus Kovačić & Šanda, 2016, had escaped attention until 2016, when it was discovered. Due to its overall superficial morphological similarity, G. incognitus used to be confused with a much rarer species, Gobius bucchichi Steindachner, 1870, which was considered one of the most common shallow-water gobies in the Mediterranean Sea. In this work, we tested the suitability of the genetic data (mitochondrial gene encoding cytochrome b) for identifying and distinguishing between these two goby species, and assessed the congruency between the distribution records based on genetic data and those based on morphological identification. We analysed material of 304 specimens of G. incognitus and G. bucchichi from 49 localities covering a considerable part of the Mediterranean Sea, Black Sea, and the Atlantic Ocean near Gibraltar, representing 19 geographically well-separated areas. We detected 270 sequences of G. incognitus, and only 34 of G. bucchichi. In both species, a high haplotype variability was observed. The sequence species identity matched morphological identification for all specimens for which vouchers were available. The mean uncorrected p-distance between G. incognitus and G. bucchichi was 13%, while the mean intraspecific distances were much lower (0.63% and 0.68%, respectively). We found 79 fixed mutations between these two species. Data on distribution based on genetic identification are completely congruent with published results based on morphological identification. The results of this study support molecular methods as a reliable tool for distinguishing morphologically similar fish species, which is particularly useful when only tissue is available for determination. [ABSTRACT FROM AUTHOR]