Previous laboratory studies of the Brown Anole, Anolis sagrei, have shown that males distinguish between familiar female cage mates and unfamiliar females in mating tests and preferentially mate with unfamiliar females. The purpose of this study was to test the hypothesis that males distinguish between former female residents of their territories and unfamiliar, nonresident females as preferred mating partners. To test the hypothesis, a total of 36 pairs of reproductively active males and females were captured from a natural habitat in the Miami, Florida area. For half of the pairs, a male and a female resident of the male's territory were captured, and for the other half, a male and a nonresident female unfamiliar to the male were captured. Within five hours of capture, the male and female of each pair were placed into a test cage in the laboratory and videotaped for one hour. Males paired with unfamiliar, nonresident females copulated significantly more frequently than males paired with former female residents of their territories. Moreover, the proportion of males that copulated was significantly greater in males that were paired with unfamiliar females than in males that were paired with resident females. These results support the hypothesis that male A. sagrei distinguish between former female residents of their territories and unfamiliar, nonresident females as preferred mating partners and suggest that free-living males might exhibit a mating preference for unfamiliar females over female residents of their territories.