Metapopulation-structured species can be negatively affected when landscape fragmentation impairs connectivity. We investigated the effects of urbanization on genetic diversity and gene flow for two sympatric amphibian species, spotted salamanders (Ambystoma maculatum) and wood frogs (Lithobates sylvaticus), across a large (>35,000 km2) landscape in Maine, USA, containing numerous natural and anthropogenic gradients. Isolation by distance (IBD) patterns differed between the species. Spotted salamanders showed a linear and relatively high variance relationship between genetic and geographic distance (r = 0.057; p < 0.001); whereas, wood frogs exhibited a strongly non-linear and lower variance relationship (r = 0.429; p < 0.001). Scale dependence analysis of IBD found gene flow has its most predictable influence (strongest IBD correlations) at distances up to 9 km for spotted salamanders and up to 6 km for wood frogs. Estimated effective migration surfaces revealed contrasting patterns of high and low genetic diversity and gene flow between the two species. Population isolation, quantified as the mean IBD residuals for each population, was associated with local urbanization and less genetic diversity in both species. The influence of geographic proximity and urbanization on population connectivity was further supported by distance-based redundancy analysis and multiple matrix regression with randomization. Resistance surface modelling found interpopulation connectivity to be influenced by developed land cover, light roads, interstates, and topography for both species, plus secondary roads and rivers for wood frogs. Our results highlight the influence of anthropogenic landscape features within the context of natural features and broad spatial genetic patterns, in turn supporting the premise that while urbanization significantly restricts interpopulation connectivity for wood frogs and spotted salamanders, specific landscape elements have unique effects on these two sympatric species.