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Kawthoolei (defined as all Karen National Union administrative areas and encompassing Karen State, Myanmar), a global hotspot for biodiversity, is increasingly under threat due to deforestation, mining, human population growth, indiscriminate burning, and infrastructure development. Pinpointing biodiversity hotspots to focus conservation efforts is integral given the limited funding and capacity of local conservation actors to undertake activities throughout Kawthoolei. We obtained wildlife presence data for 23 medium-large mammal species of conservation concern from 7 years of camera trapping efforts throughout Kawthoolei and produced species distribution models, which were used to prioritize areas for conservation action. Presence only data were combined with bioclimatic, topographical, anthropogenic, land cover, and biotic interaction variables to produce Maxent models covering 87,612 km2. Species specific maps were weighted according to their IUCN Red List of Threatened Species status and combined to pinpoint areas with high biodiversity, weighing areas higher that harbored Vulnerable and Endangered species. Individual species were predicted present in 1.44–32.17 % of the study area. Priority maps classified 55.91 % of the total study area as having no species present, 25.07 % as low priority, 10.73 % as medium priority, and 8.29 % as high priority. The protected area system currently covers 26.8 % of the high priority habitats available. While most endemic species continue to persist in Kawthoolei, models demonstrated a scattered distribution of high priority areas throughout Kawthoolei, which may lack connectivity. Additional conservation actions that supplement the current protected area system may be necessary to curb extirpation risks, including the demarcation and active patrolling of additional protected areas in high priority landscapes and activities that promote connectivity between high-quality habitats.