We attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has a high genetic barrier to HIV-1 development of resistance to DRV. We conducted selection using a mixture of 8 highly multi-protease inhibitor (PI)-resistant, DRV-susceptible clinical HIV-1 variants (HIV-1_MIX) containing 9 to 14 PI resistance-associated amino acid substitutions in protease. HIV-1_MIX became highly resistant to DRV, with a 50% effective concentration (EC₅₀) ∼333-fold greater than that against HIV-1_NL4-3. HIV-1_MIX at passage 51 (HIV-1_MIX^P51) replicated well in the presence of 5 μM DRV and contained 14 mutations. HIV-1_MIX^P51 was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1_MIX^P51 had a resemblance with HIV-1_C of the HIV-1_MIX population, and selection using HIV-1_C was also performed; however, its DRV resistance acquisition was substantially delayed. The H219Q and I223V substitutions in Gag, lacking in HIV-1_C^P51, likely contributed to conferring a replication advantage on HIV-1_MIX^P51 by reducing intravirion cyclophilin A content. HIV-1_MIX^P51 apparently acquired the substitutions from another HIV-1 strain(s) of HIV-1_MIX through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV resistance when a variety of PI-resistant HIV-1 strains are generated, as seen in patients experiencing sequential PI failure, and ensuing homologous recombination takes place. HIV-1_MIX^P51 should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.