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Reference conditions are difficult to find in the Anthropocene but essential for effective biodiversity conservation. Aquatic ecosystems in the Andes‐Amazon transition zone of Colombia are now at high risk due to expanded human activities after peace agreements in 2016 ended armed conflict because lands formerly controlled by FARC and other armed groups are now prone to agricultural and urban expansion. Particularly, expanding human land use may reduce fish diversity across the altitudinal gradient, especially in the premontane streams (i.e., <500 m a.s.l.) because lands are more amenable to human use than at greater altitudes. We evaluated fish α‐diversity (measured as species richness, total abundance, and effective species number) and β‐diversity (spatial and temporal) in 12 sites over 8 years bracketing the end of armed conflict. All α‐diversity and β‐diversity analyses were evaluated relative to categorical altitude (< or >500 m) and continuous altitude. Strong differences in fish community structure among sites occurred as a function of altitude. Fish communities exhibit altitudinal biodiversity gradients that are consistent in space and time, and that need to be accounted for conservation and management considerations. Our results provide a reference to identify short‐ and long‐term changes due to impending human land use at a critical moment for the conservation of tropical fish diversity. Similar studies in other areas of the upper Amazon Basin are needed to evaluate effects of subsequent human activities on diversity patterns and our study area to compare to reference conditions reported here. [ABSTRACT FROM AUTHOR]