Studies on hybrid zones are essential to understand the origin and evolution of reproductive barriers in plants. To achieve this goal, multidisciplinary approaches are often required to investigate the role of multiple reproductive isolation (RI) mechanisms. For Epidendrum denticulatum and E. fulgens, two Neotropical food-deceptive orchid species, we used molecular, cytogenetic and morphological analyses, experimental crosses and environmental envelope models to assess the strength of the RI and the mechanisms that prevent species collapse when hybridization occurs. Based on genetic assignment tests, hybrids between E. denticulatum and E. fulgens were detected. However, the low frequency of hybrid specimens found, coupled with the high morphological differentiation between parental species, suggested that strong barriers exist to interspecific gene exchange. Indeed, hybrid plants were largely sterile, as determined by meiotic data and crossing experiments. In the hybrid zone studied here, strong postzygotic barriers maintain species integrity, and these RI mechanisms may be also important during early stages of speciation.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Instituto de Botânica, Núcleo de Pesquisa do Orquidário do Estado, São Paulo, SP, 04301-902, BrazilDepartamento de Ciencias Naturales, Universidad Técnica Particular de Loja, San Cayetano Alto s/n, CP 11 01 608, Loja, Ecuador.Departamento de Botânica, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rio Claro, SP, 13506-900, BrazilDepartamento de Genética, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Botucatu, SP, 18618-970, Brazil.FAPESP: 2009/15052-0FAPESP: 2010/16353-1FAPESP: 2011/22215-3FAPESP: 2012/22077-2