Reciprocal molecular markers were developed to distinguish the closely related parasitoid species Encarsia formosa Gahan and E. luteola Howard. E. formosa is widely used in the biological control of whiteflies and yet, based upon morphology, it is extremely difficult to distinguish from E. luteola. The D2 expansion region of 28S rDNA was sequenced from seven strains of E. formosa and two strains of E. luteola to assess the amount of genetic variation within and between species at this locus. From parsimony analysis we find that populations of E. formosa and E. luteola each form a monophyletic clade and are probably each other’s most closely related sister taxon. Based upon the sequence variation between species, we present a simple molecular assay to rapidly and unambiguously distinguish E. formosa and E. luteola.