Morphological and habitat similarities among the five species of the terrestrial orchid genus Calopogon have led to nomenclatural and taxonomic confusion. The taxa are marked by subtle character differences and little apparent reproductive isolation. Here we investigate allozyme diversity at the species level and the partitioning of genetic variation within and among species and their populations. Genetic identities are used to define species boundaries and suggest phylogenetic relationships. All five species of Calopogon maintain high levels of allozyme variation within their populations (P = 50.0%−94.4%, AP = 2.67–3.32, H_e = 0.11–0.43). Calopogon oklahomensis, an autotetraploid that appears to have undergone gene silencing at 13 of its 19 polymorphic loci, consistently had the highest genetic diversity values. Calopogon multiflorus, which has the most restricted range and rarest occurrence, had the lowest mean genetic diversity values. In C. oklahomensis, C. pallidus and C. tuberosus most of the genetic variation exists within rather than among populations (G_ST = 0.037–0.085). The UPGMA phenogram generated using genetic identity data has three phenetic groups and supports designation of the taxa as separate species. The data suggest that C. oklahomensis most closely resembles the basal extant taxon within Calopogon.