Background: World-wide, the notable expansion of HIV/AIDS treatment programs in resource-limited settings has lead to an increasing number of patients in need of second-line cART. To adequately address and prepare for this scenario, critical assessments of the outcomes of second-line cART are particularly relevant in settings where monitoring strategies may be inadequate. We evaluated virologic outcomes of second-line combination antiretroviral therapy (cART) among HIV-infected individuals from Brazil. Methods: This study was conducted at the Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz, at Rio de Janeiro, Brazio. For this study we included all patients who started first-line and second-line cART between 2000 and 2013. Second-line cART required a switch in the anchor drug of first-line cART. We evaluated time from second-line start to virologic failure and factors associated with increased risk of failure using multivariable Cox proportional hazards regression models. Results: Among the 1,311 patients who started first-line cART a total of 386 patients (29.5%) initiated second-line cART, out of which 35.0% and 60.6% switched from their first-line to their second-line cART when their HIV RNA was undetectable and after documented virologic failure, respectively. At second line cART initiation, median age was 38 years [interquartile range (IQR): 31-45years]. Median CD4 count was significantly different for patients starting second-line cART undetectable [412 cells/mm3 (IQR: 240-617)] compared to those starting second-line cART after documented virologic failure [230 cells/mm3 (IQR: 118-322.5)] (p < 0.01). Median time from second-line cART initiation to failure was also significantly different for patients starting second-line cART undetectable compared to those who with documented virologic failure (log-rank test p < 0.01). Multivariable Cox models showed that younger age, lower education, and HIV RNA level were independently associated with an increased hazard of second-line failure among those with documented virologic failure at start of second-line cART. Conclusions: We have shown that in a middle-income country with universal access to cART, having a detectable HIV RNA at the start of second-line cART as well as younger age and lower education negatively impact second-line outcomes. Our findings could guide HIV treatment efforts as to which strategies would help maximize the durability of these regimens.