Severe emaciation and mortalities, suggestive of mycobacterial infections, were recently reported in both adult and young wild red deer (Cervus elaphus) in the south-eastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis (Mbo), due to gross and microscopic similarities with lesions caused by Mycobacterium avium subspecies paratuberculosis (Map) or Mycobacterium avium subspecies avium (Maa). The aim of this study was to improve the molecular species-specific identification of Mbo, Maa, and Map in deer mycobacterial infections. DNA banding patterns were assessed prior and after Hpy188I restriction of f57:usptream (us)-p34 duplex amplicons. As reported, the duplex f57:us-p34 PCR duplex differentiated Mbo from Map and Maa infections, whereas the restriction step differentiated single Map and Maa from mixed Map/Maa infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 138 upstream the us-p34 initiation codon in all Maa strains tested, regardless of their origin and the IS901PCR results. In contrast, the restriction site was abrogated in all Map strains tested, regardless of their origin, the Mycobactin J dependency and the IS900 PCR results. Consequently, the two-step strategy, i.e. duplex us-p34:f57 PCR and Hpy188I restriction, allowed to exclude Mbo infection and to identify single (Map and Maa) or mixed (Map/Maa) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of Map, the absence of the Hpy188I restriction site in the us-p34 amplicon.