Identifying spatial patterns is important for the conservation and management of fish diversity in streams. The spatial variation along the upstream-downstream gradient in stream fish assemblages has been substantially investigated previously. However, most investigations have focused on the species-composition-based taxonomic organizations but the species-trait-based functional organizations have received less attention. In this study, we sampled fishes from 27 segments along the upstream-downstream gradient of the Xin'an River during May and October 2017. Our aims were to examine the longitudinal variation in taxonomic and functional assemblages of stream fish assemblages, and to determine the relative importance of local habitat, segment spatial position, and landscape condition on fish assemblage composition. According to the feeding habits and locomotion traits of the fishes, the 44 species collected in this study were divided into four feeding groups, five locomotion groups, and 14 combined feeding-locomotion groups. A two-way crossed ANOSIM showed that both taxonomic and functional organizations differed significantly among the 1^st- to 5^th-order streams, but they did not differ significantly between May and October. According to SIMPER, the spatial patterns of abundance-dominant species and functional guilds were similar along the upstream-downstream gradient. From the 1^st- to 3^rd-order streams, the changes in fish assemblages agreed with the patterns of community nestedness, i.e., small assemblages nested within larger assemblages. However, from the 3^rd- to 5^th-order streams, the changes agreed with patterns of community turnover, i.e., both species/guild loss and gain occurring from one assemblage to another. Variance partitioning showed that all three types of explanatory variables measured (i.e., local habitat, landscape condition, and segment spatial position) explained 33.6% of the spatial variation in taxonomic organizations and 38.5% of the functional organizations. In addition, the taxonomic organizations was significantly affected by the local habitat and segment spatial position; whereas, the landscape condition was less important. The functional organizations were significantly influenced by local habitat and landscape condition, but the segment spatial position variables were less important. Overall, our results suggest that, along the upstream-downstream gradient, both taxonomic and functional organizations of fish assemblages exhibit similar levels of spatial variation. However, spatial variation in taxonomic organizations is driven by both environmental filtering and species dispersal, whereas the functional organizations are mainly determined by environmental filtering.