We tested the utility of molecular markers for distinguishing between two closely related species, Gonatocerus morrilli (Howard) and Gonatocerus walkerjonesi S. Triapitsyn (Hymenoptera: Mymaridae), to evaluate whether postrelease G. morrilli specimens could be discriminated in the field. Initially, postrelease specimens from California collected in 2002 and 2003 were analyzed. Amplification size of the internal transcribed spacer (ITS) region 2 demonstrated that all of the specimens were of the G. walkerjonesi ITS2 genotype. Inter-simple sequence repeat-polymerase chain reaction DNA fingerprinting of specimens from the original G. morrilli“release” colony demonstrated that the DNA banding pattern was superimposable to that of G. walkerjonesi, confirming that the G. morrilli colony was contaminated. A new G. morrilli colony was initiated in spring 2005, and we continued to survey random postrelease specimens from the 2004–2006 collections. As expected, from 2004 and most of 2005, only the G. walkerjonesi ITS2 genotype was detected. In fall 2005 and in the spring and fall 2006, we detected the G. morrilli ITS2 genotype at sites where the new colony was previously released. Analyses with two newly developed “one-step” species-specific ITS2 diagnostic markers were in accordance with the results of the markers described above, demonstrating the usefulness of the former studies of natural enemy establishment in biological control programs.