Coreius heterodon is an important commercial fish species in the upper and middle reaches of Yangtze River, and one of the appropriate fishes for the study of possible ecological effects of the Three Gorges Dam. However, the museum C. heterodon specimens were preserved in formalin. It is necessary to approach the methods of effectively extracting the genomic DNA from formalin-fixed C. heterodon, and the feasibility of using the extracted DNA in microsatellite- and mitochondrial analyses. In the present paper, the genomic DNA of ten formalin-fixed C. heterodon specimens was successfully extracted by the steps of removing the formaldehyde through improved gradual dehydration first and extracting the genomic DNA by the traditional phenol-chloroform method then. After primers designed, the PCR amplification was carried out, and a 205 bp of the control region of mtDNA from all of the ten specimens was successfully amplified. SSCP analysis indicated that no polymorphism was detected in this short fragment, and SSR also showed monomorphic. It was suggested that by using alcohol treatment, the formalin-fixed specimens of C. heterodon could be used as the potentially suitable biological materials in the studies of the possible changes of genetic diversity after large hydropower projects were constructed.