show Abstracthide AbstractTropical mountains hold more biodiversity than their temperate counterparts, and this disparity is often associated with the latitudinal climatic gradient that impacts the thermal niches, degree of habitat specialization, and dispersal ability of species. However, distinguishing the impact of latitude versus the background effects of species history and trait divergence is challenging due to the evolutionary distance between tropical and temperate assemblages. Here, we test whether microevolutionary processes, occurring within species, are linked to environmental variation across a sharp latitudinal transition in 21 montane birds of tropical and subtropical southern Atlantic Forest in Brazil. We found that individuals progressively occupy lower elevations and are less genetically structured following a gradient where seasonality increases with latitude. Effective dispersal within populations in the tropical mountains was lower and genomic differentiation was better predicted by the current environmental complexity of the region than within the subtropical populations, where differentiation was explained by the geographic distance among individuals. The concordant response of multiple co-occurring populations was consistent with spatial climatic variability as a major process driving distinct patterns of population differentiation, and support the idea that valley passes are strong barriers to gene flow for populations occurring in tropical mountains. Our results provide evidence for how a narrow latitudinal gradient can shape microevolutionary processes and contribute to broader scale biodiversity patterns.