Objective To investigate distinctive features in drug-resistant mutations(DRMs) and interpretations for reverse transcriptase inhibitors(RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients. Methods Forty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184 I and M230 I were more prevalent in proviral DNA than in viral RNA(Fisher’s exact test, P<0.05). Considering ‘majority resistant variants’, 15 samples(19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ‘minority resistant variants’, 22 samples(28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens. Objective To investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients. Methods Forty-three HIV- 1-infected individuals receiving first- line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. The mutations M184 I and M230 I were more prevalent in proviral DNA than in viral RNA (Fisher’s exact test, P <0.05) Considering ’total resistant variants’, 15 samples (19.48%) showed differences in drug resistance intent rpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ’minority resistant variants’, 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. the Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.