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There is increasing evidence that naturally occurring HIV-1 genetic diversity can have an impact on drug resistance. Recently, our previous study has demonstrated the natural presence of the V179D and K103R/V179D mutations associated with resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs) in HIV-1 CRF65_cpx strains. The aim of this study is to investigate the presence of natural drug-resistance mutations (DRMs) in other HIV-1 subtypes or CRFs circulating in China. A total of 14,403 pol sequences from China were retrieved from the Los Alamos HIV Sequence Database, 10,041 of which were treatment naïve and presented substantial genetic diversity. Besides the natural presence of V179D and K103R/V179D in CRF65_cpx, the natural presence of V179E was found in CRF55_01B. In all but one of the 228 patients infected with CRF55_01B, NNRTI resistance mutation V179E was present and the combination of V179E and E138G was detected in 14 treatment-naïve patients, with a rate of 6.2%. A significant trend for increasing prevalence of E138G mutation in CRF55_01B strains over time was observed (p <.001). Phylogenetic analysis was conducted to clarify the epidemiological relationship of CRF55_01B strains. Most of the sequences containing E138G mutation scattered in the big CRF55_01B cluster, which indicated the rising prevalence of E138G was mainly due to multiple mutation events rather than local transmission clusters of a particular variant containing E138G mutation. Our findings highlight the importance of molecular surveillance of CRF55_01B strains and the urgent need for implementation of effective preventive measures to reduce the transmission of CRF55_01B. • Natural presence of V179E was found in CRF55_01B. • Increasing prevalence of E138G in CRF55_01B over time was observed. • The rising prevalence of E138G was mainly due to multiple mutation events. [ABSTRACT FROM AUTHOR]