Islatravir (ISL) was a nucleoside reverse transcriptase translocation inhibitor (NRTTI) that blocked reverse transcriptase (RT) translocation on the primer: template, inhibiting HIV reverse transcription. ISL was being developed for HIV-1 infection treatment. Resistance selection studies with wild-type (WT) subtype A, B, and C viruses were done to enhance the knowledge of viral variations that might confer reduced sensitivity to ISL. No further alterations were found in the virus with a single known resistance-associated mutation in RT (K65R, L74I, V90I, M184I, or M184V). Antiviral activity testing was undertaken to investigate the impact of variations in selection studies. Only the single-codon alterations M184I and M184V lowered susceptibility by more than 2-fold compared to WT. A114S was an emergent mutation that lowered susceptibility by more than 2-fold when paired with other substitutions. Viruses harboring A114S and M184V did not reproduce in primary blood mononuclear cells (PBMCs), indicating that the combination was uncommon in clinical samples. While A114S made you less susceptible to ISL, it made you more susceptible to authorized nucleoside reverse transcriptase inhibitors (NRTIs). A114S had a distinct influence on ISL, an NRTTI, than NRTIs was likely due to the various modes of action. Compared to approved NRTIs, the outcomes show that ISL had a high barrier to resistance and a distinct mechanism.