Horizontal starch gel electrophoresis was employed to investigate allozyme variation at 19 isozyme loci, in nine closely-related Atlantic-Mediterranean gobiid fish species, to assess genetic relationships, and to compare this with a phyletic hypothesis based on morphological apomorphies. The species examined were Gobius niger, G . auratus, G. cruentatus, G. paganellus, Mauligobius maderensis, Zosterisessor ophiocephalus, Thorogobius ephippiatus, Padogobius martensii and P . nigricans. Various phenetic and cladistic analyses were performed on isozyme and morphological data. The phenetic and cladistic results from morphological data, and the cladistic results from isozyme data, were largely comparable, with P . martensii and P. nigricans forming a sister group to all the other taxa, species of Cobius forming a crown group within the latter, and stem lines formed by Mauligobius, Zosterisessor and Thorogobius, respectively. The isozymic estimates of genetic distance differed from these chiefly in the relative positions of Zosterisessor and G. auratus, which appear less similar to other species of Gohius. G. auratus shows many alleles which are unique within the genus Gobius; possible explanations for this are put forward. The position of nigricans as congeneric with Padogobius martensii is confirmed by both phenetic and cladistic analyses of isozyme data.