The biodiversity and structure of fish communities is an important indicator of anthropogenic and environmental impacts. Here, we reported the results of a survey carried out in 2010, in which we sampled the fish at twelve stations throughout the headstream region of the Dongjiang River. A total of 74 species in 7 orders, 18 families and 56 genera were collected and identified. Of these 74 species, 71 species were native while only Neosalanx tangkahkeii, Tilapia zillii and Gambusia affinis were exotic species. Among all the species, order Cypriniformes was the most dominant component (77.5%), followed by order Siluriformes (9.9%), order Perciformes (8.4%). As the results of relative abundance, the dominant species were Zacco platypus, accounted for 14.87%, followed by Opsariichthys bidens (9.93%)and Acrossocheilus parallens (9.36%). Temporal and spatial variations were also conducted fish community similarity analyses. The evaluation of fish diversity in the study area was made based on the combined information of G-F index, Shannon-Wiener index, Pielou’s evenness index, Margalef species richness index and Simpson index. Fish community structure in headstream region was dominated by omnivore, sedentary, mid & superstratum and benthic species, which is similar with that of the lower reach. However, the similarity index of fishes between headstream and lower reach was only 0.39. The major adverse impacts on the fish biodiversity in the headstream included dam construction, water pollution, sand excavation, excessive catching and harmful fishing gear.