Post-growth annealing and electron beam irradiation during cathodoluminescence were used to determine the chemical origin of the main optical emission lines in moderately and heavily Mg-doped GaN. The 3.27 eV donor-acceptor pair (DAP) emission line that dominates the emission spectrum in moderately Mg-doped (p-type) GaN was found to be strongly reduced by electron irradiation and of different chemical origin than the DAP at a similar energetic position in Si-doped (n-type) GaN. These results suggest that the acceptor responsible for the 3.27 eV DAP emission in Mg-doped GaN is Mg and that the donor (20–30 meV) is hydrogen-related, possibly a (VN-H) complex. This complex is dissociated either by electron irradiation or thermal annealing in N₂ or O₂ atmosphere. We found that upon electron irradiation, a deeper emission line (centered at 3.14 eV) emerged, which was assigned to a DAP consisting of the same Mg acceptor level and a deeper donor (100–200 meV) with a similar capture cross section as the donor in the 3.27 eV emission. Moreover, two different deep donor levels at 350±30 and 440±40meV were identified as being responsible for the blue band (2.8–3.0 eV) in heavily Mg-doped GaN. The donor level at 350±30meV was strongly affected by electron irradiation and attributed to a H-related defect.