In this study, morphological analysis, Fourier analysis of otolith and fragments of mitochondrial DNA (mtDNA) were used to detect the species differences between Acanthogobius ommaturus and A. flavimanus. The results showed that there was no overlap among the number of second dorsal fin spines, the number of second dorsal fin rays, the number of gluteal fin spines, the number of gluteal fin rays and the number of vertebrae. The results of the principal component analysis showed that the contribution rate of the first principal component was 52.101%, which mainly reflected the proportion of the body shape of the fish. The contribution rate related to the characteristics of the rear part of the trunk was the highest. The comprehensive discrimination accuracy based on the discriminant function was 100%. One-Way Analysis of variance showed that there was no significant difference between the head front indexes (such as the length of snout and the length of the upper jaw) and the indexes related to the length of the tail of two species. There are significant differences in other indicators except for the difference between them. The results of the Fourier analysis of otoliths were similar to those of the measurement characteristics analysis. The result of principal component analysis showed that the contribution rate of the first principal component was 12.181% which mainly reflected the shape characteristics of the dorsal and anterior and rear edges of otolith. The discrimination accuracy based on the discriminant function was 100%. The four fragment sequences of D-loop, COⅠ, 16S rRNA and 12S rRNA in the mtDNA of the two species were compared and analyzed. The result showed that the genetic distance between species was significantly higher than that within species based on the K-2P model. The phylogenetic tree constructed by combining the homologous sequences of other gobies in Acanthogobius downloaded from Genbank showed that the two species were clustered respectively. And the genetic difference was significant.