Abstract: Utilizing the sequence differences of the cytochrome b (Cytb) gene in mitochondrial DNA allows for the rapid and accurate identification of the externally similar Tribolium castaneum (Herbst) and Tribolium confusum Jacquelin du Val, providing an essential foundation for improving precision control of stored grain pests. This study amplified, sequenced, and analyzed the Cytb gene fragments of adult and larval stages of T. castaneum and. T. confusum, respectively. The species were identified based on differences in restriction enzyme sites of these gene fragments. The amplified gene fragments of both pest species were 640 bp in length. The sequence homology of this gene fragment between the two species was 77.19%, with 83 nucleotide transitions and 63 transversions, resulting in a transition/transversion ratio of 1.3. Five restriction enzymes (BstX I, Sal I, Xba I, Vsp I, and Age I) showed differences in the gene fragment between the two species. The digestion profiles of BstX I, Sal I, Xba I, and Vsp I were validated, and their patterns matched the analysis results. These restriction enzyme digestion profiles enable rapid and accurate identification of T. castaneum and T. confusum at both adult and larval stages. The PCR-RFLP method can accurately identify the larvae and adults of T. castaneum and T. confusum, providing scientific support for precise pest control.