The sixteenth-century Florida borderlands provide a unique setting for evaluating gender and its meanings to colonizers of the Americas. Despite being explored by European conquistadors much earlier than other Western locales, the peninsula and its hinterlands generated few riches and served as the site for no substantial settlements until the late eighteenth century. This situation differed significantly from settlements in Mexico, Peru, New England, or Virginia, the centerpieces of most studies evaluating gender in the New World. Between 1527 and 1567, multiple Spanish and French expeditions to Florida produced ample documentation of European men’s distinct impressions of women, which aligned closely with their own ethnicities, and used these depictions to explain explorer successes and failures in the region. Conclusions reached in this evaluation of conquistador conceptualizations of women and gender in the Florida borderlands both validate existing paradigms and promote new perspectives regarding the Americas as a whole.