The WHO-recommended second-line antiretroviral therapy of a pharmacologically enhanced protease inhibitor plus nucleoside or nucleotide reverse transcriptase inhibitors might be compromised by resistance. Results of the 96 week SECOND-LINE randomized trial showed that NtRTI-sparing antiretroviral therapy with LPV/r and RAL provided non-inferior efficacy to LPV/r and two or three NtRTIs in participants with virological failure of a first-line regimen of a NNRTI plus two NtRTIs. This exploratory analysis assessed the relation of baseline virological resistance with virological failure and emergent resistant on study. In the SECOND-LINE trial, selection of NtRTI as part of the second-line antiretroviral therapy was made by either genotype at local laboratory or algorithm. Genotypic resistance for the entire cohort at baseline was assessed on stored samples at a central laboratory. Virological failure was defined as plasma viral load > 200 copies/mL. Baseline viral isolates were assigned genotypic sensitivity scores by use of the Stanford HIV database version 6.3.1: a global genotypic sensitivity scores (gGSS), defined as the combined GSS for the 7 NtRTIs and a specific genotypic sensitivity scores (sGSS) defined as the genotypic sensitivity scores for the ART regimen initiated by a specific participant. Emergent resistance was reported on samples with a viral load > 500 copies/mL. A multivariate logistic regression with backward elimination assessed predictors of virological failure and emergent resistance. Of the 271 patients included in the NtRTI group and 270 in the RAL group, 215 and 236, respectively, had available baseline sequence data, and 240 and 255, respectively, had viral load measurements at 96 weeks. Median (IQR) gGSS was 3.0 (1.3-4.3) in the NtRTI group and 3.0 (1.0-4.3) in the RAL group. The median sGSS in the NtRTI group was 1.0 (0.5-1.8). Multivariate analysis showed significant associations between virological failure and less than complete adherence at week 4 (odds ratio 2.18, 95% CI 1.07-4.47, p=0.03) and week 48 (2.49, 1.09-5.69, p=0.03), baseline plasma viral load > 100,000 copies/mL (3.43, 1.70-6.94, p=0.0006), baseline gGSS > 4.25 (4.73, 1.94-11.6, p=0.0007), and being Hispanic (3.13, 1.21-8.13, p=0.02) or African (3.49, 1.68-7.28, p=0.0008) rather than Asian. Emergence of resistance was associated with the RAL group (odds ratio 2.47, 95% CI 1.02-5.99, p=0.05), baseline viral load (1.83, 1.12-2.97, p=0.02), and absence of the K65R or K70E mutation at baseline (3.18, 1.12-9.02, p=0.03). Poor adherence was a major determinant of virological failure in participants on second-line antiretroviral therapy. In settings with limited resources, investment in optimisation of adherence rather than implementation of drug resistance testing might be advisable .