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The aim of genetically comparing different populations of the same species of natural enemies is to identify the strain that is most adapted to the environment where it will be released. In the present study, Inter-Simple Sequence Repeat-Polymerase Chain Reaction (ISSR–PCR) was utilized to estimate the population genetic structure of Gonatocerus ashmeadi (Girault) (Hymenoptera: Mymaridae), the predominant egg parasitoid of Homalodisca coagulata (Say) (Homoptera:Cicadellidae), the glassy-winged sharpshooter. Six populations from throughout the U.S. and a population from Argentina identified as near G. ashmeadi were analyzed. Four populations (California; San Antonio, Texas; Weslaco, Texas [WTX-2]; and Florida) were field collected and two (Louisiana and Weslaco, Texas [WTX-1]) were reared. Three ISSR–PCR reactions were pooled to generate 41 polymorphic markers among the six U.S. populations. Nei's expected heterozygosity values (h), including the reared population from Louisiana, were high (9.01–14.3%) for all populations, except for a reared population from WTX-1 (2.9%). The total genetic diversity value (Ht) for the field populations was high (23%). Interestingly, the Florida population that was collected from one egg mass (siblings) generated the greatest number of polymorphic markers (20) and was observed with the highest gene diversity value (14.3%). All populations, except WTX-2 generated population-specific markers. Comparison of genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated good agreement between GST and θ values, 0.38 and 0.50, respectively for field populations, and 0.44 and 0.50, respectively for all populations. Genetic divergence (D) indicated that the WTX-1 population was the most differentiated. Average D results from the Argentina population support the taxonomic data that it is a different species. The present results estimate the population genetic structure of G. ashmeadi, demonstrating genetic divergence and restricted gene flow (Nm = 0.83) among populations. These results are of interest to the Pierce's disease/glassy-winged sharpshooter biological control program because the key to successful biological control may not be in another species, but instead in different geographic races or biotypes.