To study the genetic and phylogenetic relationship of gobioid fishes in China, the representatives of 10 gobioid fishes from 2 subfamilies in China were examined by amplified fragment length polymorphism (AFLP) analysis. We established 220 AFLP bands for 45 individuals from the 10 species, and the percentage of polymorphic bands was 100%. The percentage of polymorphic loci within species ranged from 3.61 to 58.56%. Chaeturichthys stigmatias showed the greatest percentage of polymorphic loci (58.56%), the highest Nei’s genetic diversity (0.2421 ± 0.2190) and Shannon’s information index (0.3506 ± 0.3092), while Pterogobius zacalles showed the lowest percentage polymorphic loci (3.61%), the lowest Nei’s genetic diversity (0.0150 ± 0.0778) and lowest Shannon’s information index (0.0219 ± 0.1136). The topology of UPGMA tree showed that the individuals from the same species clustered together and the 10 species formed two major clades. One clade consisted Cryptocentrus filifer, P. zacalles, Tridentiger trigonocephalus, Chaeturichthys hexanema, C. stigmatias, Acanthogobius flavimanus and Synechogobius ommaturus, and the other clade consisted Odontamblyopus rubicundus, Trypauchen vagina and Ctenotrypauchen microcephalus. The results agreed with the traditional taxonomy of the morphological characters. AFLP fingerprints were successfully used to study the phylogenetic relationship of the gobioid fishes and it identified species origins of morphologically similar taxa.Key words: Phylogenetic, amplified fragment length polymorphism (AFLP), gobiidae, Amblyopinae, gobiinae