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Identifying and clarifying how stream fish assemblage patterns vary spatially and temporally are basic measures for the conservation and management of fish species. Based on data collected from 24 wadeable reaches within the Qiupu Headwaters National Wetland Park between May and October 2012, we examined the spatial and temporal patterns of the assemblage structures and diversities, collecting a total of 29 fish species belonging to four orders and ten families. The results of our survey showed influences of local habitat and tributary spatial position variables on fish assemblages. Fish diversity showed significant variations across stream-orders and seasons, which were higher in the second-order streams than in first-order streams and higher in October than in May. Habitat factors such as substrate coarseness and heterogeneity, water temperature and water depth, as well as tributary position factor-link, showed significant effects on fish diversity. Fish assemblages fitted the nested pattern that upstream assemblages presented as a nested subset of downstream assemblages. Fish assemblage structures did not vary significantly across seasons but did across stream-orders; fish assemblages between first- and second-order streams showed significant differences despite some overlap. These spatial differences mainly resulted from spatial variations of the relative abundance of Cobitis rarus, Ctenogobius sp., Zacco platypus, Phoxinus oxycephalus, Rhodeus ocellatus and Vanmanenia stenosoma, among which P. oxycephalus had higher abundance in first-order than in second-order streams but the other five species were more abundant in second-order streams. Fish assemblage structures were significantly related to substrate heterogeneity, water depth, stream order, link and C-link.