This study sought to determine the dominant circulating human immunodeficiency virus type 1 (HIV-1) subtype and associated drug resistance mutations in Ghana.This cross-sectional study was conducted with archived samples collected from patients who received care at 2 hospitals in Ghana from 2014 to 2016. Blood samples were earlier processed into plasma and peripheral blood mononuclear cells and stored at −80 °C. Ribonucleic acid (RNA) was extracted from the archived plasma. Two HIV-1 genes; protease and reverse transcriptase, were amplified, sequenced using gene-specific primers and analyzed for subtype and drug resistance mutations using the Stanford HIV Database.Of 16 patient samples successfully sequenced, we identified the predominance of HIV-1 subtypeCRF02_AG(11/16, 68%). SubtypesG(2/16, 13%), dualCRF02_AG/G(2/16, 13%), andCRF01_AE(1/16, 6%) were also observed. Major nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations,M184I/V,D67N,T215F, andK70R/Ewere found. Non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations,K103N,Y181C,V90I,F227L, andV106Awere also prevalent. Additionally, and at a lower level, protease inhibitor (PI)-resistance mutations,M46I,I54 V,V82A,L90 M, andI471 V, were also present in the sequences from antiretroviral therapy (ART)-experienced individuals. Two NRTI-associated drug resistance mutations (DRMs) (D67NandT69N) were present in sequences from 1 ART-naive individual.HIV-1 subtype CRF02_AG was most frequently detected in this study thus confirming earlier reports of dominance of this subtype in the West-African sub-region and Ghana in particular. The detection of these drug resistance mutations in individuals on first-line regimen composed of NRTI and NNRTI is an indication of prolonged drug exposure without viral load monitoring. Routine viral load monitoring is necessary for early detection of virologic failure and drug resistance testing will inform appropriate choice of regimens for such patients.