Heterogeneity in habitat often influences how organisms traverse the landscape matrix that connects populations. Understanding landscape connectivity is important to determine the ecological processes that influence those movements, which lead to evolutionary change due to gene flow. Here, we used landscape genetics and statistical models to evaluate hypotheses that could explain isolation among locations of the threatened Mojave desert tortoise (Gopherus agassizii). Within a causal modeling framework, we investigated three factors that can influence landscape connectivity: geographic distance, barriers to dispersal, and landscape friction. A statistical model of habitat suitability for the Mojave desert tortoise, based on topography, vegetation, and climate variables, was used as a proxy for landscape friction and barriers to dispersal. We quantified landscape friction with least-cost distances and with resistance distances among sampling locations. A set of diagnostic partial Mantel tests statistically separated the hypotheses of potential causes of genetic isolation. The best-supported model varied depending upon how landscape friction was quantified. Patterns of genetic structure were related to a combination of geographic distance and barriers as defined by least-cost distances, suggesting that mountain ranges and extremely low-elevation valleys influence connectivity at the regional scale beyond the tortoises’ ability to disperse. However, geographic distance was the only influence detected using resistance distances, which we attributed to fundamental differences between the two ways of quantifying friction. Landscape friction, as we measured it, did not influence the observed patterns of genetic distances using either quantification. Barriers and distance may be more valuable predictors of observed population structure for species like the desert tortoise, which has high dispersal capability and a long generation time.