The brown citrus aphid, Toxoptera citricida (Kirkaldy), is an important pest of Florida citriculture because it causes feeding damage to citrus and vectors citrus tristeza virus. Parasitoids recovered from brown citrus aphids in Florida include Lysiphlebus testaceipes (Cresson), Lipolexis scutellaris Mackauer, and Aphelinus gossypii Timberlake. Monitoring the levels of parasitism caused by each species is difficult because the parasitoids must be reared out or dissected from the aphid hosts. A simple and quick molecular approach was developed to detect and distinguish these parasitoids developing within the host aphid. Total genomic DNA was extracted from the brown citrus aphid and each of the three parasitoids and the 18S rRNA gene of each species was amplified by polymerase chain reaction (PCR). The PCR products were sequenced to obtain complete gene sequences for each species. The variable regions V2 of the genes were used to design species-specific primers for detecting and differentiating the three parasitoids. The species-specific PCR amplifications discriminated the parasitoid DNAs from each other and from the host DNA. Detection of L. testaceipes DNA within the host aphid was possible in 8% of samples during the first 2 h after parasitoid oviposition; in 66% of samples after 24 h; in 94% of samples after 48 h; and in 100% of samples after 72 h. The PCR approach described in this study provides earlier and more precise detection of parasitism and determination of species than rearing or dissection methods.