HIV-1 uses the coreceptors CCR5 and/or CXCR4 for cell entry. Monotropic CCR5-using variants are found early in the infection while CXCR4-using variants may appear after progression to AIDS. CXCR4 use may consist of both monotropic and dualtropic viruses. The viral phenotype is important in evaluating the response to CCR5 inhibitors, a new class of antiviral drugs. The coreceptor use of HIV-1 was investigated using population sequencing in 24 patients from Botswana, carrying HIV-1 subtype C and failing antiretroviral treatment, while 26 treatment-naive patients acted as controls. Single genome sequencing was used to discern minor HIV-1 populations in the treatment-experienced group. The Geno2Pheno method was employed to predict the coreceptor use phenotype from HIV-1 env gp120 V3 DNA sequences. The glycan-charge model adjusted for subtype C was also used for phenotype prediction. The viral phenotype of population sequences was predicted using Geno2Pheno in 24/24 treatment-experienced patients, of whom eight (33%) were predicted to harbor CXCR4-using strains as compared to 2/26 in the treatment-naive group (p=0.03). Single genome sequencing generated 4–23 clones/patient in the treatment-experienced group. Altogether, 90/295 (31%) putative CXCR4-using clones were identified. In 10/24 (42%) treated patients at least one clone was predicted to be CXCR4-using, further increasing the amount of identified treatment-experienced patients with CXCR4 use. Although subtype C is usually associated with comparatively little CXCR4 use, the frequency of CXCR4 use in treatment-experienced patients with subtype C can be higher, which may have implications for the administration of CCR5 inhibitors in this patient group.