Resistance to the integrase strand transfer inhibitors raltegravir and elvitegravir is often due to well-identified mutations in the integrase gene. However, the situation is less clear for patients who fail dolutegravir treatment. Furthermore, most in vitro experiments to select resistance to dolutegravir have resulted in few mutations of the integrase gene. We performed an in vitro dolutegravir resistance selection experiment by using a breakthrough method. First, MT4 cells were infected with human immunodeficiency virus type 1 (HIV-1) Lai. After integration into the host cell genome, cells were washed to remove unbound virus and 500 nM dolutegravir was added to the cell medium. This high concentration of the drug was maintained throughout selection. At day 80, we detected a virus highly resistant to dolutegravir, raltegravir, and elvitegravir that remained susceptible to zidovudine. Sequencing of the virus showed no mutations in the integrase gene but highlighted the emergence of five mutations, all located in the nef region, of which four were clustered in the 3′ polypurine tract (PPT). Mutations selected in vitro by dolutegravir, located outside the integrase gene, can confer a high level of resistance to all integrase inhibitors. Thus, HIV-1 can use an alternative mechanism to develop resistance to integrase inhibitors by selecting mutations in the 3′ PPT region. Further studies are required to determine to what extent these mutations may explain virological failure during integrase inhibitor therapy.