Abstract Empirical estimates of the relative importance of different barriers to gene flow between recently diverged species are important for understanding processes of speciation. I investigated the factors contributing to reproductive isolation between Costus pulverulentus and C. scaber (Costaceae), two closely related hummingbird-pollinated understory Neotropical herbs. I studied broad-scale geographic isolation, microhabitat isolation, flowering phenology, overlap in pollinator assemblages, floral constancy by pollinators, mechanical floral isolation, pollen-pistil interactions, seed set in interspecific crosses, and postzygotic isolation (hybrid seed germination, greenhouse survival to flowering, and pollen fertility). Aside from substantial geographic isolation, I found evidence for several factors contributing to reproductive isolation in the sympatric portion of their geographic ranges, but the identity and relative strength of these factors varied depending on the direction of potential gene flow. For C. pulverulentus as the maternal parent, mechanical floral isolation was the most important factor, acting as a complete block to interspecific pollen deposition. For C. scaber as the maternal parent, microhabitat isolation, pollinator assemblage, mechanical floral isolation, and postpollination pollen-pistil incompatibility were important. Overall, prezygotic barriers were found to be strong, resulting in 100% reproductive isolation for C. pulverulentus as the maternal parent and 99.0% reproductive isolation for C. scaber as the maternal parent. Some postzygotic isolation also was identified in the F1 generation, increasing total isolation for C. scaber to 99.4%. The results suggest that ecological factors, including habitat use and plant pollinator interactions, contributed to speciation in this system and evolved before extensive intrinsic postzygotic isolation.