Disentangling the strength and importance of barriers to reproduction that arise between diverging lineages is central to our understanding of species origin and maintenance. Evidence suggests that divergent adaptation can drive the development of ecologically and genetically based barriers to reproduction. To date, the vast majority of studies investigating the importance of different barriers to reproduction in plants have focused on short-lived temperate taxa while studies of reproductive isolation in trees and tropical taxa are rare. Here we provide the first study to systematically examine multiple barriers to reproduction in an Amazonian tree, Protium subserratum (Burseraceae) with diverging lineages of soil specialist ecotypes. Using an observational approach and a hand pollination experiments, we aimed to quantify the contributions of individual prezygotic and postzygotic barriers including ecogeographic isolation, flowering phenology, pollinator assemblage, pollen adhesion, pollen germination, pollen tube growth, seed development and hybrid fitness to total reproductive isolation between the ecotypes. We were able to identify six active barriers to reproduction including ecogeographic isolation, phenological differences, differences in pollinator assemblages, differential pollen adhesion, and low levels of hybrid seed development. We demonstrate that ecologically based prezygotic barriers are particularly strong and that a combination of intrinsic and extrinsic prezygotic and postzygotic barriers are acting to maintain near complete reproductive isolation between edaphically divergent populations of the tropical tree, P. subserratum.