A tier 2 SHIV-MK38 strain was obtained after two in vivo passages of tier 1 SHIV-MK1. SHIV-MK38#818, cloned from the MK38 strain, was neutralisation-resistant, like the parental MK38 strain, to SHIV-infected monkey plasma (MP), HIV-1-infected human pooled plasma (HPP), and KD247 monoclonal antibody (mAb) (anti-V3 gp120 env). We investigated the mechanisms underlying the resistance of #818, specifically the amino acid substitutions that confer resistance to MK1. We introduced amino acid substitutions in the MK1 envelope by in vitro mutagenesis and then compared the neutralisation resistance to MP, HPP, and KD247 mAb with #818 in a neutralisation assay using TZM-bl cells. We selected 11 substitutions in the V1, V2, C2, V4, C4, and V5 regions based on the alignment of env of MK1 and #818. The neutralisation resistance of the mutant MK1s with 7 of 11 substitutions in the V1, C2, C4, and V5 regions did not change significantly. These substitutions did not alter any negative charges or N-glycans. The substitutions N169D and K187E, which added negative charges, and S190N in the V2 region of gp120 and A389T in V4, which created sites for N-glycan, conferred high neutralisation resistance. The combinations N169D+K187E, N169D+S190N, and N169D+A389T resulted in MK1 neutralisation resistance close to that of #818. The combinations without 169D were neutralisation-sensitive. Therefore, N169D is the most important substitution for neutralisation resistance. This study demonstrated that although the V3 region sequences of #818 and MK1 are the same, V3 binding antibodies cannot neutralise #818 pseudovirus. Instead, mutations in the V2 and V4 regions inhibit the neutralisation of anti-V3 antibodies. We hypothesised that 169D and 190N altered the MK1 Env conformation so that the V3 region is buried. Therefore, the V2 region may block KD247 from binding to the tip of the V3 region.