Reference conditions are difficult to find in the Anthropocene but essential for effective conservation of biodiversity. 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. Expanding human land use may reduce fish diversity across the altitudinal gradient but especially in premontane streams (i.e., <500 m a.s.l.) because lands are more amenable to human use than at greater altitudes. We evaluated natural fish diversity in twelve sites over eight years bracketing the end of armed conflict. Strong differences in community structure (measured as species richness, total abundance, and effective species number, and multivariate analyses) occurred as a function of altitude, as measured by. Our results provide a baseline to identify short-term and long-term changes due to impending human land use at a critical moment for the conservation of tropical fish diversity.