The spatial-temporal patterns of fish assemblages in lotic systems can provide useful information in developing effective conservation measures. This study aimed to explore the spatiotemporal changes in fish assemblage and their association with environmental factors in mountain streams of Ren River, southwest China. Filed investigations were conducted at 18 sites during rainy and dry season in 2017. A total of 21 species, belonged to 3 orders, 8 families and 19 genera, were collected. Analysis of similarities (ANOSIM) showed fish assemblages structure varied significantly at the spatial scale, but not at the temporal scale. In low order sites, fish assemblages were mainly dominated by cold water and rheophilic species (e.g. Rhynchocypris oxycephalus, Scaphesthes macrolepis, Metahomaloptera omeiensis and Gnathopogon herzensteini), while those in high order sites were predominated by warm water and eurytopicity or stagnophilic species (e.g. Squalidus argentatus, Hemiculter leucisculus and Zacco platypus). Canonical correspondence analysis (CCA) showed fish assemblages were structured by a combination of large-scale landscape factors (e.g. altitude and C-link) and small-scale habitat features (e.g. channel width, water temperature and depth). Among these factors, landscape factors had the greatest influence on fish assemblage, while local habitat measures played less important roles or just acted in certain season.