Certain populations of feral or wild European Honey Bees (EHB), Apis mellifera, have developed a tolerance of or persistence against the parasitic mite Varroadestructor in the United States. Although the grooming or mite-biting behavior is a trait bees use to defend against parasitic Varroa mites, little information is available concerning the grooming or mite-biting behavior of feral EHBs. Accordingly, we observed damaged mites of feral bees in Ohio (Apis melliferaligustica) and commercial bees in four fieldseasons, 2020 to 2023, as well as feral bees in Kentucky (A. melliferamellifera) in 2023, for a total of 7494 mites. When the mite-biting behavior was compared among these three populations, feral bees of Ohio (A. m. ligustica) and Kentucky (A. m.mellifera) displayed a significantly higher percentage (22% and 27% more) than the commercial bees. To investigate the mechanism of the mite-biting behavior, we examined bee mandibles. Mandibles are the primary mouthparts and tools used toremove or amputate the Varroa mites. Gentle Africanized Honey Bees (gAHB, A. mellifera scutellata-hybrid) in Puerto Rican exhibit mite resistance through microevolution on the island. Asian Honey Bees (A. cerana) are the original natural host of V. destructor. However, little has been reported on the morphometric analysis of mandibles between the two species A. cerana and A. mellifera, and between the subspecies A. m. scutellata-hybrid and A. m. ligustica. Utilizing X-ray micro-computed tomography (microCT), we then examined the three-dimensional morphology of mandibles in (1) A. cerana, (2) gAHB A. m. scutellata-hybrid, (3) Ohio feral A. m. ligustica, and (4) package or commercial colonies A. m. ligustica. Our findings revealed distinctive three-dimensional shapes of mandibles, indicating substantial variations among these populations. These variations suggest that mandible morphology has an integral role in the bee’s defensive mechanisms against parasitic mites. We also developed the first smartphone application to quantify the mite damage by applying artificial intelligence to image analysis. This research contributes valuable insights intothe prospective selection and breeding of honey bees with enhanced mite resistance, thus promoting the sustainability of apiculture.