Molecular and geological evidence indicates that the emergence of the Isthmus of Panamá influenced the historical biogeography of the Neotropics in a complex, staggered manner dating back at least 9 Myr bp. To assess the influence of Isthmus formation on the biogeography of the harlequin beetle-riding pseudoscorpion, Cordylochernes scorpioides, we analysed mitochondrial COI sequence data from 71 individuals from 13 locations in Panamá and northern South America. Parsimony and likelihood-based phylogenies identified deep divergence between South American and Panamanian clades. In contrast to low haplotype diversity in South America, the Panamanian Cordylochernes clade is comprised of three highly divergent lineages: one clade consisting predominantly of individuals from central Panamá (PAN A), and two sister clades (PAN B1 and PAN B2) of western Panamanian pseudoscorpions. Breeding experiments demonstrated a strictly maternal mode of inheritance, indicating that our analyses were not confounded by nuclear-mitochondrial pseudogenes. Haplotype diversity is striking in western Atlantic Panamá, where all three Panamanian clades can occur in a single host tree. This sympatry points to the existence of a cryptic species hybrid zone in western Panamá, a conclusion supported by interclade crosses and coalescence-based migration rates. Molecular clock estimates yield a divergence time of approximately 3 Myr between the central and western Panamanian clades. Taken together, these results are consistent with a recent model in which a transitory proto-Isthmus enabled an early wave of colonization out of South America at the close of the Miocene, followed by sea level rise, inundation of the terrestrial corridor and then a second wave of colonization that occurred when the Isthmus was completed approximately 3 Myr bp.