Atherina boyeri is a common euryhaline teleost fish in the Mediterranean and adjacent areas, which inhabits coastal and estuarine waters, including coastal lagoons and more rarely inland waters. Several recent studies have pointed the possible existence of three distinct groups or species, one lagoon/freshwater group and two `punctuated and unpunctuated on the flanks' marine groups, within an A. boyeri species complex. This study is a combined approach using otolith shape and molecular markers to better define the structure of the species in the western Mediterranean. Genetic differentiation and species delimitation among nine Atherina boyeri populations from several marine and lagoon/brakish habitat sites in Algeria, Tunisia and France were investigated using three mitochondrial (control region, Cyt b and 16S) and one nuclear markers (2nd intron of S7). For further phylogenetic and phylogeographic study, we added sequences from Genbank covering more areas (Ionian Sea, Adriatic Sea, Tyrrhenian Sea, Black Sea, Atlantic). Five groups were found. Two of them perfectly corresponded to two species already recognized Atherina presbyter and Atherina hepsetus, both living in marine waters; and three additional, including Atherina boyeri (brackish and freshwater environments) and two independent groups of marine punctated and unpunctated individuals. Those findings are corroborated by the study of the otolith contour shape of 362 individuals of seven populations from different habitats using Fourier analysis. Individuals could be discriminated into five groups based on the first two functions (Wilk's lambda = 0.07, p \textless 0.001). Samples from Ziama inlet, marine punctuated individuals and unpunctuated marine specimens from Annaba's Gulf formed three well separated groups. Specimens from Mellah and Mauguio lagoons formed another group. The last one includes individuals from Bizerte and Thau lagoons. The divergences between them strongly support the potential species within the A. boyeri species complex. (C) 2016 Elsevier Ltd. All rights reserved.