Objectives: To investigate mutations selected in viruses from HIV-2-infected patients failing a highly active antiretroviral treatment (HAART) regimen including atazanavir/ritonavir. Methods: Twenty-eight HIV-2-infected patients previously exposed to atazanavir/ritonavir and failing therapy were studied. The protease (PR) gene was amplified and sequenced, and mutations emerging under atazanavir/ritonavir selective pressure were reported. Results: The I50L mutation emerged in 4 out of 28 HIV-2-infected patients failing a HAART regimen including atazanavir/ritonavir. Besides I50L, four PR mutations previously associated with protease inhibitor resistance (I54L, I64V, V71I and I82F) and six PR mutations of unknown impact (V10I, E37D, S43T, K45R, I75V and F85L) in HIV-2 were also identified in this small group of patients. Conclusions: Several mutations were associated with virological failure of a regimen including atazanavir/ritonavir in HIV-2-infected patients, including I50L for the first time. It should be included in HIV-2 algorithms for interpretation of genotypic resistance data, and taken into account when making therapeutic decisions for HIV-2-infected patients.