Hybridization between alien plant species and their native congeners represents a major global threat to native floras. In Hawai'i, two introduced cotton species, Pima cotton (G. barbadense) and Upland cotton (G. hirsutum), have naturalized on the main Hawaiian Islands. Both species are capable of producing fertile hybrids with the native Hawaiian cotton (G. tomentosum). Hybrid populations between Pima and Hawaiian cotton were documented at Nanakuli on the Wai'anae coast of O'ahu in 1964 by the late Stanley G. Stephens. Extant populations of Pima and Hawaiian cotton in the vicinity of those documented by Stephens (1964) were screened using both morphological and molecular (microsatellite markers) techniques to assess persistence of the original hybrid population and potential gene flow that may have occurred between the two cotton species. We did not relocate the original hybrid populations described in Stephens (1964), although a herbarium voucher that was verified as a hybrid indicates that at least one hybrid plant occurred at a nearby site until as late as 1980. No hybrids between Pima and Hawaiian cotton were found in populations of either species, suggesting that no recent gene flow has taken place and that hybrid plants have not persisted or spread.