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Forest fragmentation studies are now urgent due to increased rates of deforestation and forest fires worldwide. In South America, the bee-pollinated Cariniana estrellensis is one of the largest trees, and a paradigm for the health and sustainability of forest biomes. For a large-yet-fragmented population (four subpopulations) in the transition zone between Brazilian Savannah and Atlantic Forest, we carried out a study of pollen flow, mating system and spatial genetic structure using nine microsatellite loci. This revealed that the subpopulations are not reproductively isolated because of pollen flow from outside the study area (18.3%) and between subpopulations (16.1–31.3%). Pollen dispersal reached long distances (3.5 km), but mating occurred predominantly between larger-diameter trees located close to mother-trees. We found that C. estrellensis is self-compatible with reproduction mediated mainly by outcrossing (> 0.95), but matings were not random due to biparental inbreeding (tr: 0.048–0.124) and correlated-paternity (rp: 0.16–0.28), which was higher within (rpw: 0.524–0.95) than among fruits (rpa: 0.048–0.052). Inbreeding decreased from seedlings (0.088) to adults, indicating inbreeding depression between the seedling and adult stages. Subpopulations exhibited spatial genetic structure (50–200 m), revealing a pattern of genetic dispersion of isolation-by-distance. Seeds should be harvested from trees that are > 200 m apart for successful ex-situ conservation and populations should not be isolated by more than the maximum pollen-dispersion distance observed (3.5 km) for in-situ conservation. The findings are consistent with the maximum distance that bees can disperse pollen and thereby maintain genetic connectivity between populations and resilience to population fragmentation into forest remnants. [ABSTRACT FROM AUTHOR]