To determine the feasibility of HIV genotyping at low-level viraemia (LLV) using an in-house assay in a South African population and the prevalence, as well as the clinical relevance, of drug resistance (HIVDR) in this population. We conducted an observational, retrospective, cohort study on patient samples with LLV referred for routine HIVDR testing at a public sector Johannesburg laboratory from August 2017 to October 2018. Genotyping was performed using a nested RT-PCR assay and Sanger sequencing. The genotyping success rate was evaluated for different viraemia categories. Sequences were loaded onto the Stanford HIVdb genotypic resistance tool (version 8.7) for drug resistance interpretation. Plasma samples from 159 HIV-1-infected, treatment-experienced adults with LLV (5-999 copies/mL) were analysed. The in-house assay performed well with an overall success rate of 78.6% (125/159, 95% CI 71.6-84.3). The prevalence of drug resistance mutations in the LLV cohort was 79.2% (99/125, 95% CI 71.2-85.4) with most patients (n = 109, 68.6%) on a PI-based regimen at the time of genotyping. Of 125 sequences obtained, 73.6% (92/125) had ≥1 NRTI mutation while 70.4% (88/125) had ≥1 NNRTI mutation. Major PI mutations, including M46I and V82A, were detected in 7.2% (9/125) of patients. Current South African virological failure guidelines may keep patients on failing regimens for longer than necessary. Our data suggest that genotyping at LLV is feasible and implementation could result in earlier identification and referral of patients requiring third-line regimens.