Megalobrama pellegrini is a cyprinid fish endemic to upper reaches of the Changjiang (Yangtze) River, China, which is also an important economic species in the local area. In recent years, resources of this species have decreased sharply and its conservation has drawn great attention. In the present study, we collected 120 individuals from the Longxi River, a tributary isolated from the main channel of the Changjiang River, where M. pellegrini is still relatively abundant. Using two different molecular markers, mitochondrial cytochrome b (cyt b) gene, and nuclear microsatellite (simple sequence repeat, SSR), we analyzed the genetic diversity of this isolated population. The results show that sequence genetic diversity was low (H-d : 0.290 and P-i: 0.000 77 for cyt b gene), while the SSR genetic diversity was high (H-o: 0.824 4 +/- 0.147 2, H-e: 0.823 5 +/- 0.145 1). Analysis indicated that this population had experienced a bottleneck, with inbreeding and small effective population size (around 50). Based on SSR data, relatedness analyzing revealed that the 120 samples were grouped into 10 completely independent clusters. It was inferred that the mating system of M. pellegrini was polygamy. We suggested that the low genetic diversity could be induced by the overfishing and inbreeding depression. Therefore, we suggested that the urgent conservation measures should be taken to control the overfishing and give better conditions for the fish to grow and spawn, then to restore population size.