Geographic isolation is a key factor in shaping the genetic structure of many fish species. Two sympatric species, Culter albus and C . mongolicus , are economically important fish that are widely distributed in China and have been recently used as new aquaculture species. We used the mitochondrial DNA control region (CR) as a marker to investigate the genetic structure of the two species. Phylogenetic analysis revealed two major lineages (Lineages Ⅰ and Ⅱ) that were highly consistent with geographical patts for C . albus and C . mongolicus . Based on genetic distance, the Zhujiang (Pearl) River Basin (ZRB) populations potentially represented a cryptic subspecies, which might be differentiated as the result of strict geographic isolation, an earlier diverged event, or peripheral areas. Genetic diversity analysis suggested that the Changjiang (Yangtze) River Basin (CRB) populations are located at, or near the core region of their origination, not only due to the larger population size at the CRB, but also their habitat diversity and suitability for its survival, and the genetic diversity differences among basin populations were significant for the two species. Moreover, demographic analysis indicated that the two Culter species and most populations had undergone a period of population expansion during warm interglacial periods. However, the C . albus population of Huaihe River Basin (Weishan Lake) exhibited different patts during the interglacial period, which may due to the latest diverged time of HRB and Weishan Lake located at the permafrost. Notably, the ZRB (in Songtao Reservoir) C . mongolicus population showed no genetic diversity and had a unique haplotype, which could be treated as a special gene pool for species conservation. In summary, geographic isolation is most likely responsible for the two Culter species distribution patts.