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Abstract Landscape heterogeneity can shape genetic structure and functional connectivity of populations. When this heterogeneity imposes variable costs of moving across the landscape, populations can be structured according to a pattern of “isolation by resistance” (IBR). At the same time, divergent local environmental filters can limit gene flow, creating an alternative pattern of “isolation by environment” (IBE). Here, we evaluate IBR and IBE in the insect‐pollinated, biennial plantSabatia angularis(L.) Pursh (Gentianaceae) across serpentine grasslands in the fragmented landscape of SE Pennsylvania, USA using ~4500 neutral SNP loci. Specifically, we test the extent to which radical alteration of the landscape matrix by humans has fundamentally altered the cost of movement, imprinting a pattern of IBR dictated by land use type and intensity, and the potential for IBE in relation to a gradient of heavy metal concentrations found in serpentine soil. We reveal a strong signal of IBR and a weak signal of IBE across sites, indicating the greater importance of the landscape matrix in shaping genetic structure ofS. angularispopulations in the study region. Based on Circuitscape and least cost path approaches, we find that both low‐ and high‐intensity urbanization resist gene flow by orders of magnitude greater than “natural” habitats, although resistance to low‐intensity urbanization weakens at larger spatial scales. While cropland presents a substantially lower barrier than urban development, cumulative human land use surrounding populations predicts within‐population genetic diversity and inbreeding inS. angularis. Our results emphasize the role of forest buffers and corridors in facilitating gene flow between serpentine grassland patches and averting local extinction of plant populations.