The prevalence of HIV-1 drug resistance mutations (DRMs) was determined for a cross-section of individuals (n=8654) in five centers across Vietnam (Hanoi, Hai Phong, Da Nang, Khanh Hoa, and Can Tho) between 2008 and 2009. Following serological screening for HIV infection, HIV-1 viral load was determined, using an in-house real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay. Samples with quantifiable viral loads [all either commercial sex workers (CSW) or intravenous drug users (IDU)] underwent DRM analysis. Sequences were obtained for 92 treatment-naive individuals, the majority of whom were infected with HIV-1 CRF01_AE (99%), with one instance of subtype A1 also detected. DRMs were detected in seven treatment-naive individuals (7.6%). The most common DRMs observed were M184V, V75A/M, M41L, and K65R (NRTI) and K103N, G190A, and Y181C (NNRTI). Overall, the data from this first multicenter survey of DRMs in Vietnam indicate that the problem of transmitted drug resistance is of major concern in the highest-risk groups of IDU and CSW.