The main goal of the present study was to determine the frequency of substitutions in the cleavage sites (CS) of gag gene among non-B HIV-1 isolates from Gabon. Fifty plasma specimens, collected in 2010-2011, from HIV-1-infected patients failing first-line antiretroviral (ARV) regimens (constituted of two nucleoside reverse transcriptase inhibitors+one nonnucleoside reverse transcriptase inhibitor) (n=38) and from HIV-1-infected individuals untreated with ARV (n=12) were analyzed in the gag and gag-pol cleavage sites. Compared to HXB2 reference sequence, the total median number of substitutions in gag and gag-pol CS was 10 (range, 5-18). The cleavage site p2/NC was the most variable of the four gag CS with 100% (50/50) isolates carrying at least 1 substitution (range, 1-9). The two gag-pol TFP/p6pol and p6pol/PR CS sites were also highly variable (at least one substitution, 50/50, 100% in both cases). Substitutions at position G381 (p2/NC), L449 (p1/p6gag), and K444 (TFP/p6pol) were significantly more frequent in CRF02_AG strains, compared to other non-B strains (30.4% vs. 3.7%, p=0.03; 87.0% vs. 59.3%, p=0.03; and 91.3% vs. 59.3%, p=0.01, respectively). Other non-B subtypes were significantly more likely to harbor substitutions at position N487 (p6pol) (70.4%) than CRF02_AG (39.1%) (p=0.02). In Gabon, gag and gag-pol cleavage sites were highly polymorphic in protease inhibitor-naive patients harboring non-B HIV-1 strains. In sub-Saharan Africa, further studies are definitively required to better understand the impact of gag mutations among subjects receiving second-line LPV/r-containing regimens (monotherapy or triple combinations).