Colonization of vacant habitat is a fundamental ecological process that affects the ability of species to persist and undergo range modifications in continually shifting landscapes. Thus, understanding factors that affect and limit colonization has important ecological and conservation implications. Epiphytic orchids are increasingly threatened by various factors, including anthropogenic habitat disturbance. As cleared areas (e.g. pastures) are recolonized by suitable host trees, the establishment and genetic composition of epiphytic orchid populations are likely a function of their colonization patterns. We used genetic analyses to infer the prevailing colonization pattern of the epiphytic orchid, Brassavola nodosa. Samples from three populations (i.e. individuals within a tree) from each of five pastures in the dry forest of Costa Rica were genotyped with neutral nuclear and chloroplast markers. Spatial autocorrelation and hierarchical genetic structure analyses were used to assess the relatedness of individuals within populations, among populations within pastures and among populations in different pastures. The results showed significant relatedness within populations (mean r = 0.166) and significant but lower relatedness among populations within a pasture (mean r = 0.058). Our data suggest that colonization of available habitats is by few individuals with subsequent population expansion resulting from in situ reproduction, and that individuals within a tree are not a random sample of the regional seed pool. Furthermore, populations within a pasture were likely colonized by seeds produced by founders of a neighbouring population within that pasture. These results have important ramifications for understanding conservation measures needed for this species and other epiphytic orchids.