Habitat loss and fragmentation in the Mojave Desert have been increasing, which can create barriers to movement and gene flow leading to decreased populations of native species. Disturbance and degradation of Mojave desert tortoise habitat includes linear features (e.g. highways, railways, and a network of dirt roads), urbanized areas, and their associated infrastructure, mining activities, energy distribution systems, and most recently, utility-scale solar facilities. To evaluate the spatial genetic structure of tortoises in an area experiencing rapid habitat loss, we conducted field surveys from 2015-2017 and genotyped 299 tortoises at 20 microsatellite loci within and around Ivanpah Valley along the California/Nevada border. We used a Bayesian clustering analysis to examine population genetic structure across valley and mountain pass habitat. Spatial principal components analysis was included to further investigate population genetic structure with isolation-by-distance. To explicitly incorporate landscape features (e.g. habitat and anthropogenic linear barriers) we used maximum likelihood population effects. We assessed recent gene flow on the landscape through maximum likelihood pedigree analyses of relatedness. We detected three to four genetic clusters with high levels of admixture that generally corresponded to three valleys separated by mountain ranges, and a genetically distinguishable population in one mountain pass. Pedigree analyses showed second order relationships up to 60 km apart suggesting a greater range of interactions and inter-relatedness between individuals than previously suspected. Our results support historical gene flow with isolation-by-resistance, and reveal a genetic signal indicative of reduction in genetic connectivity across two parallel linear features (a railway and a highway). This work demonstrates the value of protecting connected tracts of functional habitat and the importance of connectivity research in conservation.