Objectives Lamivudine (3 TC ) and emtricitabine ( FTC ) are considered interchangeable in recommended tenofovir disoproxil‐fumarate ( TDF )‐containing combination antiretroviral therapies ( cART s). This statement of equivalence has not been systematically studied. We compared the treatment responses to 3 TC and FTC combined with TDF in boosted protease inhibitor ( PI )‐based cART for HIV ‐1‐infected patients. Methods An observational study in the AIDS Therapy Evaluation in the Netherlands ( ATHENA ) cohort was carried out between 2002 and 2013. Virological failure rates, time to HIV RNA suppression < 400 copies/mL, and time to treatment failure were analysed using multivariable logistic regression and Cox proportional hazard models. Sensitivity analyses included propensity score‐adjusted models. Results A total of 1582 ART ‐naïve HIV ‐1‐infected patients initiated 3 TC or FTC with TDF and ritonavir‐boosted darunavir (29.6%), atazanavir (41.5%), lopinavir (27.1%) or another PI (1.8%). Week 48 virological failure rates on 3 TC and FTC were comparable (8.9% and 5.6%, respectively; P = 0.208). The multivariable adjusted odds ratio of virological failure when using 3 TC instead of FTC with TDF in PI ‐based cART was 0.75 [95% confidence interval ( CI ) 0.32–1.79; P = 0.51]. Propensity score‐adjusted models showed comparable results. The adjusted hazard ratio ( HR ) for treatment failure of 3 TC compared with FTC was 1.15 (95% CI 0.58–2.27) within 240 weeks after cART initiation. The time to two consecutive HIV RNA measurements < 400 copies/ mL within 48 weeks ( HR 0.94; 95% CI 0.78–1.16) and the time to treatment failure after suppression < 400 copies/ mL ( HR 0.94; 95% CI 0.36–2.50) were not significantly influenced by the use of 3 TC in TDF / PI ‐containing cART . Conclusions The virological responses were not significantly different in treatment‐naïve HIV ‐1‐infected patients starting either 3 TC / TDF or FTC / TDF and a ritonavir‐boosted PI .