Geographic isolation in rainforest refugia and local adaptation to ecological gradients may both be important drivers of evolutionary diversification. However, their relative importance and the underlying mechanisms of these processes remain poorly understood because few empirical studies address both putative processes in a single system. A key question is to what extent is divergence in signals that are important in mate and species recognition driven by isolation in rainforest refugia or by divergent selection across ecological gradients? We studied the little greenbul, Andropadus virens, an African songbird, in Cameroon and Uganda, to determine whether refugial isolation or ecological gradients better explain existing song variation. We then tested whether song variation attributable to refugial or ecological divergence was biologically meaningful using reciprocal playback experiments to territorial males. We found that much of the existing song variation can be explained by both geographic isolation and ecological gradients, but that divergence across the gradient, and not geographic isolation, affects male response levels. These data suggest that ecologically divergent traits, independent of historical isolation during glacial cycles, can promote reproductive isolation. Our study provides further support for the importance of ecology in explaining patterns of evolutionary diversification in ecologically diverse regions of the planet.