A primary goal of molecular ecology is to understand the influence of abiotic factors on the spatial distribution of genetic variation. Features including altitudinal clines, topography and landscape characteristics affect the proportion of suitable habitat, influence dispersal patterns, and ultimately structure genetic differentiation among populations. We studied the effects of altitude and topography on genetic variation of long-toed salamanders (Ambystoma macrodactylum), a geographically widespread amphibian species throughout northwestern North America. We focused on 10 low altitude sites (< 1200 m) and 11 high-altitude sites in northwestern Montana and determined multilocus genotypes for 549 individuals using seven microsatellite loci. We tested four hypotheses: (1) gene flow is limited between high- and low-altitude sites; and, (2) gene flow is limited among high-altitude sites due to harsh habitat and extreme topographical relief between sites; (3) low-altitude sites exhibit higher among-site gene flow due to frequent flooding events and low altitudinal relief; and (4) there is a negative correlation between altitude and genetic variation. Overall FST values were moderate (0.08611; P < 0.001). Pairwise FST estimates between high and low populations and a population graphing method supported the hypothesis that low-altitude and high-altitude sites, taken together, are genetically differentiated from each other. Also as predicted, gene flow is more prominent among low-altitude sites than high-altitude sites; low-altitude sites had a significantly lower FST (0.03995; P < 0.001) than high altitude sites (FST = 0.10271; P < 0.001). Use of Bayesian analysis of population structure (BAPS) resulted in delineation of 10 genetic groups, two among low-altitude populations and eight among high-altitude populations. In addition, within high altitude populations, basin-level genetic structuring was apparent. A nonequilibrium algorithm for detecting current migration rates supported these population distinctions. Finally, we also found a significant negative correlation between genetic diversity and altitude. These results are consistent with the hypothesis that topography and altitudinal gradients shape the spatial distribution of genetic variation in a species with a broad geographical range and diverse life history. Our study sheds light on which key factors limit dispersal and ultimately species’ distributions.