The neotropical region, encompassing the tropical and subtropical regions of the Americas and extending from southern Mexico through Central America and the Caribbean to South America, is known for its high biodiversity and complex ecosystems. Natural hybridization might have played an important role in shaping such biodiversity, but studies focused on understanding its influence are still scarce when compared with other regions of the World. Yet, understanding the mechanisms and consequences of hybridization is essential for effective conservation and management of these diverse ecosystems. In this study, we aimed to detect the presence of natural hybridization between Chaetogastra longifolia (Vahl) DC. and Chaetogastra campii (Wurdack) P.J.F.Guim. & Michelang. (formerly included in Tibouchina). Previous ecological studies have suggested the possibility of gene flow between them due to the existence of individuals with conflicting morphological traits in sympatric populations where the two species occurred. To achieve this, forty-eight samples of the two species of Chaetogastra, including putative hybrids, were analysed through plastid DNA sequencing results and taxon-specific amplified fragment length polymorphism (AFLP) fragment distribution analysis. The results confirmed the hybrid nature of all putative hybrids. The AFLP patterns of most hybrids demonstrated a closer relationship to C. campii, suggesting hybridization to be asymmetric with this species more often used as a maternal progenitor of hybrids. Some individuals collected as pure were also revealed to be of hybrid origin, indicating the possibility of backcrosses with parental species. Future studies should focus on understanding the ecological and evolutionary outcomes occurring after hybridization, as well as the existence of natural hybrids between other species of Chaetogastra.