Bactrocera minax (Enderlein), commonly known as the Chinese citrus fly, is a citrus pest native to China and
nearby countries. B. minax can cause substantial losses in citrus orchards. B. minax can be spread to countries free
from it by the global trade of citrus and by travelers carrying citrus. Timely, convenient, and accurate identification
of B. minax is essential in preventing its spread. In the present study, the nuclear ribosomal internal
transcribed spacer 2 (ITS2) amplicon was used to design species-specific primer pairs that enable B. minax to be
distinguished from 11 other fruit fly species. Four forward and four reverse species-specific primers were
designed, and out of all possible sets of species-specific primer pairs obtained after intermixing them, seven sets
of species-specific primer pairs were able to accurately identify B. minax. For B. minax identification, specific
fragments ranging from 83 to 431 base pairs in length were amplified. The validity of the specific band only in
B. minax was determined by visually inspecting the gel profile of the PCR product. B. minax was correctly
identified using the designed species-specific primer pairs, with no cross-amplification with the 11 other fruit fly
species included in the experiments. In addition to saving DNA sequencing costs, the application of these speciesspecific
primer pairs facilitates rapid identification of B. minax, with identification in border scenarios being
completable within 2–3 h.