Morphometric, allozymic, and mitochondrial DNA variability previously indicated that the Mediterranean anchovy Engraulis encrasicolus includes two distinct forms, one of the inshore habitat, and the other one of the open-sea habitat. Here, we showed that the two forms significantly differ by several morphological characters. To test the hypothesis, proposed previously, that the two forms are distinct biological species, we used length-polymorphic, exon-primed intronic PCR markers from the creatine-kinase multigenic family and genetically characterized anchovy samples collected in the northwestern Mediterranean and in the eastern Atlantic. Large genetic differences were found between the two forms (Weir and Cockerham's (θ) over cap = 0.397 to 0.586). In contrast. geographic variation in the open-sea forin at the scale of the eastern Atlantic/northwestern Mediterranean was weak ((θ) over cap= -0.006 to 0.042). This again demonstrated considerable restriction to nuclear gene flow between inshore and open-sea anchovy populations. In addition to previous results from allozymes this confirmed their status as distinct biological species, namely Engraulis albidus sp. nov. and Engraulis encrasicolus L. (C) 2004 Academie des sciences. Published by Elsevier SAS. All rights reserved.