The recently described anti-human immunodeficiency virus type 1 (HIV-1) human mAb PG9 and PG16 are cross-clade broadly neutralizing. Therefore, it can be postulated that the targeted epitope(s) are highly conserved among variants of the entire group M. We analysed the sensitivity to PG9 and PG16 of pseudotyped viruses carrying envelope glycoproteins from the viral quasispecies of three HIV-1 clade CRF01_AE-infected patients. The broad heterogeneity in sensitivity to PG9 and PG16, despite closely genetically related envelope glycoproteins issued from single individuals, allowed us to identify two gp120 cross-clade conserved residues, a lysine at position 168 in the V2 loop and an isoleucine at position 215 in the C2 region, whose substitutions were associated with resistance to PG9 and PG16. By site-directed mutagenesis, we confirmed both in clades B and CRF01_AE that the substitutions K168E and I215M have a major impact on PG9 and PG16 neutralization sensitivity of pseudotyped viruses.