Abstract: Rapidly identifying juvenile individuals of Liriomyza trifolii (Burgess) from Liriomyza sativae Blanchard is crucial in plant quarantine. We report a molecular method to identify L. trifolii based on real-time polymerase chain reaction (PCR). By comparing partial DNA sequences of mitochondrial COI genes of L. trifolii samples collected from Guangdong and Taiwan provinces in China, Japan, Philippine, Israel, Germany, the USA, Mexico and Honduras sequenced by authors, and those of related species recorded in GenBank, a L. trifolii-specific probe was developed. There was no difference in individuals of different stages tested by this probe. The total time for real-time PCR assay system was 2 h, and it would save 3–7 h compared with conventional PCR.