Citation: L opez-Delgado, E. O., K. O. Winemiller, and F. A. Villa-Navarro. 2020. Local environmental factors influence beta-diversity patterns of tropical fish assemblages more than spatial factors. Ecology 101(2):Abstract. A major goal in ecology is to understand mechanisms that influence patterns of biodiversity and community assembly at various spatial and temporal scales. Understanding how community composition is created and maintained also is critical for natural resource management and biological conservation. In this study, we investigated environmental and spatial factors influencing beta diversity of local fish assemblages along the longitudinal gradient of a nearly pristine Neotropical river in the Colombian Llanos. Standardized surveys were conducted during the low-water season at 34 sites within the Bita River Basin. Physical, chemical, and landscape parameters were recorded at each site, and asymmetric eigenvector maps were used as spatial variables. To examine the relative influence of dispersal and environmental variables on beta diversity and its components, distance-based redundancy analysis (db-RDA) and variation partitioning analysis were conducted. We proposed that spatial scale of analysis and position within the river network would constrain patterns of beta diversity in different ways. However, results indicated that in this system, high beta diversity was consistent among species assemblages no matter the scale of analysis or position within the river network. Species replacement (turnover) dominated beta diversity, an indication of the importance of species sorting. These findings suggested that conservation of fish diversity in tropical rivers requires maintenance of both habitat heterogeneity (spatial variation in habitat conditions) and connectivity at the scale of entire river basins.