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Aim Conserving stream biota could require strategies that preserve habitats conveying resistance to ecological impacts of changing land use and climate. Retrospective analyses of species' responses to anthropogenic disturbances can inform such strategies. We developed a hierarchical framework to contrast environmental conditions underlying persistence versus extirpation of an imperilled stream fish, Candy Darter (Etheostoma osburni), over decades of changing land use. The decline of E. osburni may broadly represent the challenge of conserving sensitive freshwater species in intensively used upland environments. Location New River drainage, Appalachian Mountains, USA. Methods We surveyed fish and habitat in historically occupied sites to identify population refugia, and used multivariate and spatial analyses to address three questions: (a) what are the environmental correlates of refugia? (b) are the pathways by which land use impacts instream habitat constrained by catchment- and/or segment-scale features? and (c) are E. osburni distributional dynamics spatially structured and explained by fine sediment and warm stream temperatures? Results We confirmed a recently localized distribution similar to other upland species, marked by at least seven extirpations from streams throughout E. osburni's southern range. Catchment-scale features primarily constrained land use and finer-scale habitat, leading to either extirpations or population-supporting refugia defined by features at multiple scales. Refugium habitats contained cooler temperatures and less fine sediment. Rare mismatches between persistence and habitat suitability were explained by network location, suggesting unmeasured environmental gradients and/or dispersal contributed to distributional dynamics. Main conclusions We provided insight at multiple spatial scales into how aquatic species' distributions become fragmented and localized. Our results demonstrate that natural landscape heterogeneity imparts spatially variable resistance of sensitive species to intensive land uses. By recognizing the scale-specific features that buffer populations from extirpation, conservation strategies could be tailored to protect naturally occurring refugium habitats and focus restoration in systems where such habitats are broadly lacking.