Abstract: The genetic diversity of fish is seriously inferred from human disturbance factors such as water pollution and habitat destruction. Recent studies on the genetic diversity of fishes in small and medium-sized rivers in eastern China have focused on simple species, but there has been less comprehensive evaluation of fish genetic diversity and its causes. In this study, Cao’e River was selected as the representation of middle and small rivers in East China to evaluate the general genetic diversities of its fishes using mitochondrial cytochrome b gene (Cyt b). The results showed that the haplotype diversity indices of Cyt b from the 21 species and 26 population were ranged from 0.074 to 0.987, and their nucleotide diversity indices were ranged from 0.00019 to 0.00520. The genetic diversities among different species were large. Comparing the genetic diversities of fishes in different sections of Cao’s River, the haplotype diversity indices of fishes decreased gradually from the estuary to upstream (P<0.05). The haplotype diversity indices in species populations from Cao’e River were significantly lower than that of same species from large rivers such as the Yangtze River and Yellow River (P<0.05). The haplotype diversity indices of sensitive fishes were significantly lower than those of middle tolerance fishes (P<0.05). Both the haplotype diversity indices and nucleotide diversity indices of three species, Pseudorasbora parva, Pelteobagrus nitidus and Mastacembelus aculeatus, in the populations from the upstream sampling site of Jinling were lower than those in the populations of same species from the sampling site of middle and lower reaches, Xianyan. This result implied that the overall genetic diversities of fishes from Cao’e River lied in middle or even low level, and that water pollution and overfishing might be the mainly reasons to reduce the genetic level of Cao’e River. In summary, our results provided an important theoretical basis for the management, protection, exploitation and utilization of fish resource of Cao’e River, and even of the middle and small rivers in East China.