Decisions regarding landscape management, restoration, and land acquisition typically depend on land managers’ interpretation of how wildlife selects habitat. Such assessments are particularly important for umbrella species like the endangered Florida panther (Puma concolor coryi), whose survival requires vast wildlands. Some interpretations of habitat selection by panthers have been criticized for using only morning locations in defining habitat use. We assessed habitat selection using a Euclidean distance analysis (EDA) and location data collected throughout the diel period from GPS collars deployed on 20 independent Florida panthers. We corroborated aspects of earlier analyses by demonstrating selection of forested habitats by panthers. We also confirmed selection of open habitats (i.e., marsh–shrub–swamps, prairie–grasslands), a novel result. Habitat selection did not vary by sex or season but varied by time of day. Panthers were located closer to wetland forests in the daytime and used prairie–grasslands more at night. Our assessment of the effect of patch size on selection of forest habitat revealed that panthers were not solely reliant on large patches (> 500 ha) but utilized patches of all sizes (≤ 1 ha, > 5–10 ha, > 1000 ha, etc.). Our results emphasize the importance of collecting panther location data throughout the diel period when assessing habitat selection. Conservation strategies for panthers should consider a mosaic of habitats, a methodology that will protect other sensitive flora and fauna in South Florida.