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Copepods, present in a wide range of water bodies, are an important component of freshwater ecosystems and their biodiversity has been much studied in marine and freshwater ecosystems. However, no previous genetic data are available that allow an assessment of population-genetic diversity and differentiation of the copepod Sinocalanus tenellus from Chinese freshwaters. We analyzed DNA sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene from eleven S. tenellus populations (individual lakes) from China, ten of which exhibited a high genetic diversity. Low to high population differentiation was detected among the populations. Interestingly, substantial genetic divergence was detected between WLS (Wuliangsu, in Inner Mongolia) and other locations, indicating the presence of two lineages of S. tenellus in East Asia. Moreover, we found that two distinct clades of S. tenellus were separated by the reference “S. sinensis” clade, suggesting they were parts of a complex of cryptic species of S. tenellus. This study will contribute to an understanding of the diversity and biogeography of copepods in freshwater ecosystem in China.