Bactrocera tsuneonis and Bactrocera minax are the most destructive pests that damage citrus in China. These key pests hinder the citrus trade, cause significant financial losses, drastically lower citrus production and quality, and decrease farmer enthusiasm for citrus planting. Bactrocera minax and B. tsuneonis are very similar in all life stages. There are limited morphological characteristics to differentiate the adult species, and it is nearly impossible to differentiate these two species in the egg and larval stages. Loop-mediated isothermal amplification (LAMP) is a rapid and robust diagnostic tool used to identify these two species accurately. We designed two sets of primers to distinguish B. minax and B. tsuneonis using DNA barcoding region of the COI gene. Only 50 min was needed under a constant temperature of 65°C to determine the species of the two flies. The reaction system has high specificity and sensitivity, in which these two species can be accurately distinguished between different geographical populations and 1.0 ng/µL was the lowest DNA concentration that could be detected. Our primers can quickly identify these key pests without knowing their morphology, which could facilitate plant protection workers at the primary level to solve problems in plant quarantine.