Our aim was to investigate the mutations in protease (PR), reverse transcriptase (RT), and integrase (IN) gene regions in human immunodeficiency virus (HIV) using a single amplicon via next-generation sequencing (NGS). The study included plasma samples from 49 HIV-1-positive patients, which were referred for HIV-1 drug resistance testing during 2017. A nested polymerase chain reaction (PCR) was performed after the RNA extraction and one-step reverse transcription stages. The sequencing of the HIV genome in the PR, RT, and IN gene regions was carried out using MiSeq NGS technology. Sanger sequencing (SS) was used to analyze resistance mutations in the PR and RT gene regions using a ViroSeq HIV-1 Genotyping System. PCR products were analyzed with an ABI3500 GeneticAnalyzer (Applied Biosystems). Resistance mutations detected with NGS at frequencies above 20% were identical to the SS results. Resistance to at least one antiretroviral (ARV) drug was 22.4% (11 of 49) with NGS and 10.2% (5 of 49) with SS. At least one low-frequency resistance mutation was detected in 18.3% (9 of 49) of the samples. Low-frequency resistance mutations resulted in virological failure in only one patient. The cost of the analyses was reduced by sample pooling and multiplex analysis using the MiSeq system. This is the first study in Turkey to use NGS technologies for the detection of resistance mutations in all three gene (PR, RT, IN) regions using a single amplicon. Our findings suggest that NGS is more sensitive and cost-effective than the SS method.