Some of the members of the Mycobacterium avium–intracellulare (MAI) complex are recognized as human pathogens in both immunocompromised and immunocompetent patients. The current molecular methods that are available for genotyping the MAI complex members can be both expensive and technically demanding. In this report, we describe for the first time the application of a real-time PCR and high-resolution melt approach to differentiate between the complex members by targeting a member of the Pro- Pro-Glu gene family, MACPPE24. To this end, reference strains of the M. avium subspecies and Mycobacterium intracellulare were used to optimize the technique. Then, this real-time PCR–high-resolution melt approach was used to distinguish ten M. avium ssp. hominissuis field isolates from the M. intracellulare reference strain.