Abstract: In order to screen for the specific molecular markers among Oxyeleotris marmoratus, Oxyeleotris lineolatus and their hybrids, the candidate gene of sumo-interacting motif-containing protein 1（simc1） was selected to distinguish the three fish species. Using the mRNA sequence of hybrids simc1 gene, P1 primer pair was designed to amplify the genome DNA of the three species of Oxyeleotris, and the 893 bp fragment was amplified. This sequence had no SNP within intraspecific individuals of the three Oxyeleotris species, but 21 SNP sites were identified among interspecific individuals of the three species of Oxyeleotris, and all of them were in exon regions, including 7 synonymous mutations and 14 nonsynonymous mutations. Then P2 and P3 primer pairs were designed for allele-specific PCR（AS-PCR） according to the genotypes of the SNP15 in the three species of Oxyeleotris. After PCR amplification and electrophoresis, O. lineolatus could be identified by a 222 bp fragment with P2 and no fragment with P3 primer pair, and O. marmoratus could be identified by no fragment with P2 and a 566 bp fragment with P3. And the hybrids could be identified by both 222 bp and 566 bp fragments with P2 and P3. The sensitivity of P2 and P3 was detected by the gradient dilution of DNA templates. The results showed that the minimum detection concentrations of genomic DNA were 5×10^-4mg/L and 5×10^-2mg/L for P2 and P3 primer pairs, respectively. The screening for specific molecular markers of the three species of Oxyeleotris and the establishment of rapid detection methods can provide technical supports for the study of germplasm resources and the breeding of new varieties.