Transmission of drug-resistant HIV (TDR) has been associated with virologic failure of "first-line," nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). A national ART program began in Peru in 2004. We evaluated the prevalence of TDR in individuals initiating ART and their virologic outcome during 2 years of ART. HIV-infected, ARV-naive subjects who met criteria to start ART in Lima, Peru were enrolled in a longitudinal observational study between July 2007 and February 2009. Blood plasma and cells obtained prior to ART initiation were assessed for antiretroviral (ARV) resistance by an oligonucleotide ligation assay (OLA) sensitive to 2% mutant at reverse transcriptase (RT) codons K103N, Y181C, G190A, and M184V and a subset by consensus sequencing. A total of 112 participants were enrolled; the mean CD4 was 134 ± 89 cells/μl and the median plasma HIV RNA was 93,556 copies/ml (IQR 62,776-291,364). Drug resistance mutations conferring high-level resistance to ARV were rare, detected in one of 96 (1%) evaluable participants. This subject had the Y181C mutation detected in both plasma and peripheral blood mononuclear cells (PBMCs) at a concentration of 100% by OLA and consensus sequencing; nevertheless nevirapine-ART suppressed her viral replication. Consensus sequencing of 37 (19%) participants revealed multiple polymorphisms that occasionally have been associated with low-level reductions in ARV susceptibility. A low prevalence of TDR was detected among Peruvians initiating ART. Given the increasing availability of ART, continuing surveillance is needed to determine if TDR increases and the mutant codons associated with virologic failure.