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Background Prevalence and factors associated with etravirine (ETV) resistance mutations among patients failing on first-generation non-nucleoside reverse transcriptase inhibitors (NNRTI) merit investigation. Methods The study comprised an analysis of all sequential patients attending the Institute of Infectious Diseases (Brescia, northern Italy) who performed a genotypic resistance testing (GRT) after ≥3 months of a stable NNRTI-based regimen between 2001 and 2006. Multivariable ordinal logistic regression analysis was performed to assess predictors of ETV resistance mutations. Results Out of 248 strains, 153 (61.7%) harboured ≥1 ETV resistance mutations. In particular, 88 (35.5%), 53 (21.4%) and 12 (4.8%) harboured one, two and three mutations, respectively. The most frequent mutations were G190A (23%), Y181C (23%) and K101E (14.1%). Use of nevirapine (odds ratio [OR] 2.73; 95% confidence level [CI] 1.62–4.62; P500 copies/ml and GRT (per month, OR 1.05; 95% CI 1.01–1.09; P=0.012) were associated with a greater number of ETV resistance mutations. Conversely, higher CD4+ T-cell counts at nadir (per 100 cells/mm3, OR 0.81; 95% CI 0.67–0.98; P=0.029) and use of lamivudine/emtricitabine (OR 0.57; 95% CI 0.37–0.87; P=0.009) were protective. Accumulation of ETV resistance-associated mutations was demonstrated by sequential GRT in 4/35 patients (all treated with nevirapine). Conclusions Mutations associated with ETV resistance were common among patients failing on NNRTI, but prevalence of viral strains harbouring three mutations was low. Use of efavirenz and co-administration of lamivudine reduced the risk of ETV resistance. The continued use of the current NNRTI in a failing regimen may select for additional resistant variants.