Species-specific molecular markers were developed to identify and distinguish between two parasitoids of the brown citrus aphid, Toxoptera citricida Kirkaldy, in Florida. PCR primers were developed for Lysiphlebus testaceipes Cresson and Lipolexis oregmae Gahan (= scutellaris Mackauer) with DNA sequences from the internal transcribed spacer (ITS) region between the 5.8S and 28S nuclear rRNA genes. With High-fidelity PCR, the L. testaceipes-specific primer produced a 520-bp band while that of L. oregmae resulted in a 270-bp band. Eggs of both parasitoids within their aphid hosts could be detected by 6 h after oviposition, but 100% detection rates only occurred after 24 h. A sensitivity analysis indicated that a parasitoid egg within a single aphid could be detected 100% of the time when combined with DNA from up to 36 unparasitized aphids. A single first instar parasitoid could be detected by High-fidelity PCR when the parasitized aphid was combined with up to 500 unparasitized aphids, indicating a high level of sensitivity. Species-specific primers detected both immature parasitoid species within aphids commonly found in citrus in Florida, including Aphis craccivora Koch, Aphis gossypii Glover, Aphis spiraecola Patch, Toxoptera aurantii Boyer and T citricida. This High-fidelity PCR assay provides an efficient method to monitor establishment of L. oregmae in citrus groves in this classical biological control program in Florida. Techniques; Pathological techniques; Biochemistry; Genetics; Parasites diseases and disorders; Parasites; Insect parasites; Hosts; Insect hosts Lipolexis oregmae; Lysiphlebus testaceipes (Aphidiidae ); Genetic techniques; Diagnostic techniques; DNA fingerprinting; Role in detection of parasites in hemipteran hosts; Nucleic acids; Molecular genetics; DNA sequences; Hemipteran hosts; Toxoptera citricida Toxoptera citricida (Aphidoidea); Genetic techniques; Diagnostic techniques, use of DNA fingerprinting; Nucleic acids; Molecular genetics; DNA sequences; Hymenopteran parasites; Lipolexis oregmae & Lysiphlebus testaceipes none