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Tylenchulus semipenetrans is the most economically important and widespread nematode pest of citrus in China. rDNA-ITS of 14 populations of T. semipenetrans which were collected from different citrus groves or Chinese fir (Cunninghamia lanceolata) plantations in China were amplified and sequenced. The species-specific primers were designed for the first time to diagnosis T. semipenetrans based on the sequences of rDNA-ITS regions of geographic population above. The primers were sensitive to amplify the expected band size (297 bp) from DNA template of a single second-stage juvenile (J2) or different life stages of T. semipenetrans. No specific band was amplified from 15 non-target nematode species which were commonly found in citrus groves. Specificity and reliability of the primers were validated by further PCR amplification of 16 extra populations of T. semipenetrans collected from 4 provinces of China. The primers successfully detected a single J2 of T. semipenetrans within a whole nematode community comprising a large numbers of non-target nematode. The developed diagnostic technique can be used for accurate identification of T. semipenetrans and also as a decision tool for nematode management for citrus or Chinese fir in China.