A simple PCR-RFLP consisting in the digestion of a fragment of the 12S ribosomal RNA (12SrRNA) with species-specific restriction enzymes (MboII and HinfI) was developed to distinguish northeastern Atlantic species in the genus Atherina. Unambiguous identification of morphologically similar sympatric species is frequently difficult, especially when younger specimens are included in the analysis. Genetic and morphological analysis of a total of 123 atherinids collected near the mouth of the Tagus estuary (west Portugal) confirmed that only Atherina presbyter (sand smelt) adults and juveniles are recurrently using these marine rocky habitats as spawning and nursery grounds, respectively. The importance of applying non-destructive methods is emphasized. In the future, the conditions involved in the transition from marine A. presbyter to estuarine/riverine A. boyeri should be further evaluated along with the ability of larvae and juveniles to cope with river flow and tidal currents drift.