Riparian wetlands are complex ecosystems containing species diversity that may easily be affected by anthropogenic disturbances. Preble's meadow jumping mouse (Zapus hudsonius preblei) is a federally threatened subspecies dependent upon riparian wetlands along the Front Range of Colorado and southeastern Wyoming, USA. Although habitat improvements for Preble's meadow jumping mouse are designed at multiple spatial scales, most knowledge about its habitat requirements has been described at a landscape scale. Our objective was to improve our understanding of Preble's meadow jumping mouse microhabitat characteristics within high-use areas (hotspots), which are essential for the development of effective management and conservation strategies. We evaluated Preble's meadow jumping mouse habitat by describing areas of high use and no use as determined from monitoring radiocollared individuals. A comparison of microhabitat characteristics from random samples of high-use and no-use areas indicated that mice use areas closer to the center of the creek bed and positively associated with shrub, grass, and woody debris cover. Distance to center of the creek bed, and percent of shrub and grass cover also had the greatest relative importance of habitat variables modeled when describing high-use areas. High-use areas contained 3 times more grass cover than forb cover, and overall had a greater proportion of wetland shrub and grass cover. However, proportion of cover type (shrub or grass) did not vary greatly between high-use and no-use areas. Our results suggest that management and conservation efforts should continue to focus on establishment of native wetland vegetation near streams and creeks. For example, vegetation should include shrubs such as willow (Salix spp.), narrowleaf cottonwood (Populus angustifolia), alder (Alnus incana), grasses such as fescue (Fescue spp.), sedges (Carex spp.), and rush (Juncus spp).