Lessepsian species are marine organisms that enter the Mediterranean through the Suez Canal, a phenomenon that has dramatically increased in recent decades. The present study describes the morphological characteristics of the sagittal otoliths of 22 Lessepsian fish species collected at four locations in the Mediterranean Sea. These structures are commonly used in the identification of species found in the digestive tracts of predators, and their morphological description is mainly needed as a tool for trophic studies. Here we used the Automated Taxon Identification (ATI) system of the AFORO web database to determine the accuracy of classifying Lessepsian fish otoliths compared with otoliths of native Mediterranean species. The otolith contour analysis correctly classified 92.5% of the specimens, showing that these species have otoliths that can be clearly distinguished from native ones. Four different groups of otoliths were identified according to the morphological differences between the otoliths and the ATI analysis results: a) unusual shapes with no similarities to native species; b) unusual shapes with similarities to phylogenetically distant native species; c) common shapes with similarities to phylogenetically close native species; and d) common shapes with morphological characters related to other native congeners, although they are not classified with them.