Over the past 20 years, the 51% reduction in the number of deaths due to the human immunodeficiency virus (HIV) that causes acquired immunodeficiency syndrome (AIDS) pushed these infections down to 19th place in 2019 among the world’s top causes of death since 2010 [
1]. In 2022, HIV remained a major public health problem worldwide, having caused 40.4 million deaths since 1981, and its transmission continues in all countries [
2]. In that year, the Joint United Nations Programme on HIV/AIDS (UNAIDS) estimated the number of people living with HIV (PLHIV) worldwide at 39.0 million, with two-thirds (25.6 million) living in sub-Saharan Africa. It also reported that, in the same year, 29.8 million people around the world were receiving antiretroviral treatment [
2], that 630 000 PLHIV died of HIV-related causes, and 1.3 million new HIV infections occurred [
2]. Resistance to antiretrovirals can compromise their effectiveness in treating HIV, leading to an increase in the number of infections, morbidity, and mortality associated with HIV [
3,
4]. Nevertheless, access to antiretroviral therapy (ART) has improved considerably over the past decade [
5]. With the ever-increasing number of PLHIV receiving antiretroviral therapy and longer treatment durations, failures of first-line treatments are increasingly being reported. Recently, 21 surveys carried on resistance linked to nevirapine (NVP) or efavirenz (EFV) showed 10% resistance among PLHIV who started first-line antiretroviral therapy. That leads healthcare workers to prescribe second-line ART treatments for PLHIV [
6]. As a result, resistance to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class of antiretrovirals is up to three times more frequent in people who have already been exposed to antiretrovirals [
7]. The frequency of failure of first-line antiretroviral therapy for HIV infection in adults is estimated at between 20% and 82% [
8]. In contrast, virological failure rates in adults on second-line regimens have been reported to range from 8 to 41% in resource-limited countries [
9]. One futuristic goal that has been set is that four million patients will be receiving second-line antiretroviral treatment in sub-Saharan Africa by 2030 [
10]. In 2016, WHO guidelines for HIV treatment recommended a second-line regimen consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) and a ritonavir-boosted protease inhibitor (PI) [
11]. The global action plan on HIV resistance to antiretroviral molecules (HIVDR) builds on the sustainable development global commitment presented in the new Agenda 2030 to end the AIDS epidemic by 2030 [
12]. WHO guidelines recommend the systematic monitoring of viral load (VL) and the extension of HIV resistance testing [
11,
13,
14]. For the biological monitoring of PLHIV, Gabon, through its National Programme for the Control of Sexually Transmitted Infections (PNLIST), adheres to WHO guidelines, which recommend that all HIV-positive people begin antiretroviral treatment (ART) [
15]. Between 2002 and 2021, three studies on resistance mutations were carried out on patients in Gabon. First of all in Libreville in 2002, Vergne et al. reported 58% of resistance mutations [
16]. Then in Franceville in 2012, Caron et al. and in 2021 Engone-Ondo et al. found 56.7% and 21.9% of resistance mutations, respectively [
17‐
18]. However, all these studies, although focused on the protease gene, were carried out on small samples (19) or in semi-rural regions that did not cover the whole country and failed to provide a comprehensive view of the diversity of HIV antiretroviral resistance mutations related to protease inhibitors in Gabon. The aim of this study, therefore, was to investigate HIV antiretroviral resistance mutation profiles in the protease gene in PLHIV receiving second-line ARVs treatment in Gabon.