Background: The HPTN 083 trial demonstrated that long-acting cabotegravir (CAB-LA) was superior to tenofovir-disoproxil fumarate/emtricitabine for HIV pre-exposure prophylaxis (PrEP). Integrase strand transfer inhibitor (INSTI) resistance-associated mutations (RAMs) were detected in some participants with HIV infection. We used a low viral load INSTI genotyping assay to evaluate the timing of emergence of INSTI RAMs and assessed whether HIV screening with a sensitive RNA assay would have detected HIV infection before INSTI resistance emerged. Methods: Single-genome sequencing to detect INSTI RAMs was performed for samples with viral loads <500 copies/mL from five participants with previously-identified INSTI RAMs and two with no prior genotyping results. Results: Major INSTI RAMs were detected in all seven cases. HIV RNA testing identified infection before major INSTI RAMs emerged in four cases and before additional major INSTI RAMs accumulated in one case. Most INSTI RAMs were detected early when the viral load was low and CAB concentration was high. Conclusions: When using CAB-LA PrEP, earlier detection of HIV infection with a sensitive RNA assay may allow for earlier treatment initiation with the potential to reduce INSTI resistance risk. Further studies are needed to evaluate the value and feasibility of HIV RNA testing with CAB-LA PrEP.