HIV-1 group M is classified into nine different subtypes. Most antiretroviral (ARV) drugs have been developed for subtype B, and the response of non-B subtypes in terms of susceptibility and the acquisition of drug resistance when facing those drugs is largely unknown. In this study, we aimed to address differences in the impact of protease inhibitor (PI)-selected mutations on subtypes B and G. ARV-treated, HIV-positive patients regularly monitored at the Hospital de Egas Moniz, in Lisbon, Portugal, were examined for the presence of PI-associated primary mutations (301 subtype B and 184 subtype G), and for the selection of those mutations over the time of PI exposure. Forty-three subtype G patients were phenotyped for susceptibility to all available PIs through VIRCO's Antivirogram (R), and compared with a similar dataset of subtype B patients. Mutation I54V/L was selected by nelfinavir in subtype G isolates, a mutation not previously described for this drug in subtype B. L90M was associated with a lower reduction in the susceptibility of subtype G to nelfinavir when compared with subtype B, and with no reduced susceptibility to saquinavir. This was compensated for by the acquisition of M89I in subtype G. L90M did not reduce the susceptibility of subtype G to saquinavir, in contrast to subtype B. Likewise, the pattern I54V/L-L90M did not reduce the susceptibility of subtype G to indinavir and saquinavir. Indinavir-associated mutations M46I/L, I84V and V82A/F/T developed earlier in subtype B across the time of exposure to that drug when compared with subtype G counterparts. Our results provide proof of principle and support the growing evidence that subtype-specific responses to ARVs exist. Data presented here highlight inconsistencies in current genotyping interpretation algorithms inadequately applied to all HIV-1 subtypes.