Madagascar’s tortoises face population declines due to human activities, including illegal trade. A potential health threat is antimicrobial resistance (AMR) genes in tortoises, which hinder treatment and signal environmental contamination. This study screens AMR genes in confiscated and protected tortoises to assess exposure differences. Findings will guide preventive measures to limit AMR spread at the wildlife-human interface, aiding conservation efforts for Madagascar’s endangered tortoises.