Phylogeographic studies provide an important framework for investigating the mechanisms operating during the earliest stages of speciation, as reproductive barriers can be examined among divergent lineages in a geographic context. We investigated the evolution of early stages of intrinsic postmating isolation among different populations and lineages of Epidendrum denticulatum, a Neotropical orchid distributed across different biomes in South America. We estimated genetic diversity and structure for both nuclear and plastid markers, using a haplotype network, differentiation tests, Bayesian assignment analysis, and divergence time estimates of the main lineages. Reproductive barriers among divergent lineages were examined by analyzing seed viability following reciprocal crossing experiments. Strong plastid phylogeographic structure was found, indicating that E. denticulatum was restricted to multiple refuges during South American forest expansion events. In contrast, significant phylogeographic structure was not found for nuclear markers, suggesting higher gene flow by pollen than by seeds. Large asymmetries in seed set were observed among different plastid genetic groups, suggesting the presence of polymorphic genic incompatibilities associated with cytonuclear interactions. Our results confirm the importance of phylogeographic studies associated with reproductive isolation experiments and suggest an important role for outbreeding depression during the early stages of lineage diversification.