Biogeographic barriers such as rivers have been shown to shape spatial patterns of biodiversity in the Amazon basin, yet relatively little is known about the distribution of genetic variation across continuous rainforest. Here, we characterize the genetic structure of the brilliant-thighed poison frog (Allobates femoralis) across an 880 km long transect along the Purus-Madeira interfluve south of the Amazon river, based on 64 individuals genotyped at 7 609 SNP loci. A population tree and clustering analyses revealed four distinct genetic groups, one of which was strongly divergent. These genetic groups were concomitant with femoral spot coloration differences, which was intermediate within a zone of admixture between two of the groups. The location of these genetic groups did not consistently correspond to current ecological transitions between major forest types. A multi-model approach to quantify the relative influence of isolation-by-distance (IBD) and isolation-by-environmental resistance (IBR) nevertheless revealed that, in addition to a strong signal of IBD, spatial genetic differentiation was explained by IBR primarily linked to dry season intensity (r 2 = 8.4%) and canopy cover (r 2 = 6.4%). We show significant phylogenetic divergence in the absence of obvious biogeographical barriers and that finer-scaled measures of genetic structure show patterns that are associated with environmental variables also known to predict the density of A. femoralis.© The American Genetic Association 2020. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.