Background: Fostemsavir belongs to the new class of attachment inhibitors (AIs); it inhibits the entry of HIV into CD4+ T-lymphocytes by blocking conformational changes in gp120. This is a promising AI, but previous phenotypic data showed that genetically divergent HIV-1 group O could present natural resistance to this drug. These data were obtained from only two strains, which are not representative of the high intra-group genetic diversity. Moreover, no data are available concerning the other divergent HIV-1 groups (N and P). Objectives: To further investigate the natural genotypic susceptibility of HIV-1 groups O, N and P (HIV-1 non-M) to fostemsavir, using a large set of sequences. Methods: The frequency of eight substitutions associated with decreased susceptibility to fostemsavir (L116P, A204D, S375M/H, M426L, M434I, M475I and V506M), was investigated in 111 gp120 sequences from groups O (n = 100), N (n = 9) and P (n = 2). Results: All HIV-1 group N sequences harboured the three substitutions S375M, M426L and M434I, whereas only 1% and 10% of HIV-1 group O sequences harboured the S375H + M426L and S375H + M434I patterns, respectively. The main genetic profile of HIV-1 groups P and O combined S375H with two atypical substitutions (M426S and M434L). Five group O sequences did not display any of the eight substitutions, but had atypical residues with unknown impact. Conclusions: The genetic polymorphisms in the gp120 of HIV-1 non-M viruses support the hypothesis that these viruses could largely be resistant to inhibition by fostemsavir. Only 5% of group O strains could display full genetic susceptibility. Extensive phenotypic studies are now required.