Melioidosis is a bacterial infection caused byBurkholderia pseudomallei(B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection ofB. pseudomalleiis crucial for preventing and controlling melioidosis. However, identifyingB. pseudomalleiis challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identifyB. pseudomalleiin less than 40 min. We analyzed 1722B. pseudomalleigenomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguishB. pseudomalleifrom its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detectB. pseudomalleiin human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification ofB. pseudomalleiin clinical and environmental samples, aiding in the prevention and control of melioidosis.