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Aims To investigate spatial congruence between ecological niches and genotype in two allopatric species of desert tortoise that are species of conservation concern. Location Mojave and Sonoran Desert ecoregions; California, Nevada, Arizona, Utah, USA. Methods We compare ecological niches of Gopherus agassizii and Gopherus morafkai using species distribution modelling (SDM) and then calibrate a pooled-taxa distribution model to explore local differences in species-environment relationships based on the spatial residuals of the pooled-taxa model. We use multiscale geographically weighted regression (MGWR) applied to those residuals to estimate local species-environment relationships that can vary across the landscape. We identify multivariate clusters in these local species-environment relationships and compare them against models of (a) a geographically based taxonomic designation for two sister species and (b) an environmental ecoregion designation, with respect to their ability to predict a genotype association index for these two species. Results We find non-identical niches for these species, with differences that span physiographic and vegetation niche dimensions. We find evidence for two distinct clusters of local species-environment relationships that when mapped, predict an index of genotype association for the two sister taxa better than did either the geographically based taxonomic designation or an environmental ecoregion designation. Main conclusions Exploring local species-environment relationships by coupling SDM and MGWR can benefit studies of biogeography and conservation. We find that niche separation in habitat selection conforms to genotypic differences between sister taxa of tortoise in a recent secondary contact zone. This result may inform decision making by agencies with regulatory or land management authority for the two sister taxa addressed here.