Urban development in the Florida Keys, USA, mandates an understanding of how habitat requirements for Florida Key deer (Odocoileus virginianus clavium) interact with vegetation changes caused by development. Our study objectives were to (1) determine Key deer habitat use at different spatial scales, (2) evaluate vegetation changes and identify vegetation types most threatened by development, and (3) provide guidelines to direct land acquisition programs in the future. We identified 6 vegetation types: pineland, hammock, developed, freshwater marsh, buttonwood, and mangrove. Key deer (n = 180; 84 F, 96 M) preferred upland vegetation types (>1 m above mean sea level; pineland, hammock, developed) and avoided tidal or lower-elevation areas (<1 m above mean sea level; freshwater marsh, buttonwood, mangrove). Analyses of Geographic Information System (GIS) coverages suggested that historical development impacted near-shore habitats while recent trends pose a greater risk to upland areas (pineland, hammock). Because uplands are preferred by Key deer, conservation measures that include land acquisition and habitat protection of these areas may be needed.