On the basis of morphoanatomical parameters, the sand smelt species (Atherina boyeri Risso, 1810) is viewed as a highly polymorphic complex. In this study, intraspecific sequence variation in a portion of the cytochrome b gene was examined in 88 individuals from Tunisia and France. The correlation between the results of statistical analysis of the sequence data using a variety of tree-building algorithms and morphoanatomical analyses demonstrated the subdivision into three putative species: A. boyeri, which only includes non-punctuated fishes, A. punctata, which corresponds to punctuated fishes and A. lagunae, which corresponds to atherines living in lagoons.