Commercial products for determining the drug-resistance mutations of HIV-1 are expensive and usually focus on a particular HIV-1 subtype. In this study, a more cost-effective in-house method was compared side-by-side with the ViroSeq™ Genotyping System 2.0 for determining resistant mutations in China's most prevalent subtype, CRF01_AE. Plasma samples were obtained from 205 patients infected with HIV-1, and subtypes were verified using the Stanford calibrated population resistance tool. The capacity for determining positive samples was not significantly different between the in-house (93.8%) and ViroSeq™ (96.5%) methods. For the clade of subtype CRF01_AE in particular, complete concordance between the methods was observed for the protease and reverse-transcriptase regions of the HIV-1 pol gene, and concordance for overall DRRMs and HLDRMs was 99.5% and 100%, respectively. Although 51 discordant mutations were found, further analysis verified that most of mutations had minimal impact on antiviral drugs. Excellent overall concordance (97.7%) was achieved for the resistance reports between the two methods for CRF01_AE. Thus, the performance and effectiveness of determining resistance-associated mutations were nearly equivalent between the cost-effective in-house method and the ViroSeq™ system, the greatly potential application of the in-house method in China for many patients infected with HIV-1 located in resource-limited regions.