The eastern spotted skunk (Spilogale putorius) is a species of conservation concern in much of its range and has experienced a decline since the early to mid‐1990s. But the subspecies that inhabits peninsular Florida, the Florida spotted skunk (S. p. ambarvalis), might still be abundant and is an important nest predator of the endangered Florida grasshopper sparrow (Ammodramus savannarum floridanus). To gain insight on this little‐studied subspecies and inform potential management strategies, we conducted a resource selection study on the Florida spotted skunk. We examined 5 hypotheses for den site selection related to den type, vegetation, and landscape characteristics in a dry prairie ecosystem in central Florida. We tracked 36 individual skunks to 757 den sites. Using discrete choice analysis, we found that male and nonbreeding female skunks at our study site were 5 times more likely to select a mammal burrow over a gopher tortoise (Gopherus polyphemus) burrow, that selection of a den site increased 34% for each 1‐burrow increase in the number of nearby burrows, and that selection of a den site increased 3% for every 10‐cm increase in a visual obstruction index. Similarly, breeding female skunks were more likely to select mammal burrows and shallow depressions over gopher tortoise burrows by 16‐fold and 13‐fold, respectively, and selection of a den site increased by 75% for every 1‐burrow increase in the number of nearby burrows. In contrast to previous studies that occurred in forested, mountainous environments elsewhere in the species' range, our findings suggest that den characteristics might be more important than landscape or vegetation characteristics to Florida spotted skunk den site selection in dry prairie. Additionally, the frequency of prescribed fires on the landscape did not appear to affect Florida spotted skunk den site selection. Thus, Florida spotted skunks in this ecosystem might be landscape generalists, thereby potentially limiting the ability of managers to control nest predation by this subspecies through habitat management. © 2019 The Wildlife Society.