In the prairies of North America, remnant native grasslands are threatened by continuing agricultural extensification. Fragmentation of the remaining grassland isolates patches and limits the potential for dispersal of native species. We explored these impacts by analyzing the spatial pattern of native grassland habitats in the Prairie Coteau region of eastern South Dakota and western Minnesota, USA. Undisturbed grasslands were mapped using a GIS database of land use history combined with manual interpretation of high-resolution aerial photographs. Network analysis based on graph theory was used to examine how connectivity changed depending on the potential movement distances of organisms and to identify important patches that made large contributions to connectivity throughout the broader network. Interpatch movement was assessed using Euclidian distance as well as cost-weighted distance that assigned lower movement cost to grasslands than to human-modified land cover types. Much of the undisturbed grassland was concentrated in a single large cluster, which was connected to other habitat concentrations via corridors of stepping stone patches. A small number of keystone patches, whose loss would have a disproportionately large effect on overall connectivity, were also identified. The locations of the major corridors were relatively consistent across different movement distances. Information about patch-level importance for overall network connectivity should be taken into account when prioritizing conservation and restoration. Future studies can build on this research by conducting more detailed assessments focused on particular species of concern and portions of the study area where connectivity is most limited.