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Background: Viral load monitoring is used as an important biomarker for diagnosing treatment failure in patients with HIV infection/AIDS. Ethiopia has started targeted viral load monitoring. However, factors leading to virological failure are not well understood and studied. Thus, the aim of this study was to identify the determinants of virological failure among HIV-infected patients on highly active antiretroviral therapy at the University of Gondar Referral Hospital, Northwest Ethiopia. Methods: A case-control study was conducted from May to June 2015. Cases were subjects who had already experienced virological failure; controls were those without virological failure. Data were extracted from 153 cases and 153 controls through chart review. A multivariate logistic regression analysis was carried out to identify factors associated with virological failure, and variables with a p-value <0.05 were considered statistically significant. Results: In this study, higher odds of virological failure was observed among patients aged <35 years (adjusted odds ratio [AOR] =2.52, 95% CI: 1.33, 4.77), who had had CD4+ count <200 cells/mm3 (AOR=9.03, 95% CI: 4.40, 18.50), showed poor adherence to antiretroviral therapy (ART) (AOR=15.80, 95% CI: 6.90, 36.50), and had taken ART for longer durations of 25-47 months (AOR=3.00, 95% CI: 1.10, 8.40) and ≥48 months (AOR=6.70, 95% CI: 2.70, 16.60). Conclusion: This study showed that patients aged <35 years and with recent low CD4 count, poor adherence to treatment, and longer exposure to ART were positively and significantly associated with virological treatment failure. Therefore, evidence-based intervention should be implemented to improve adherence to ART, which in turn helps to boost immunity (CD4) and suppresses viral replication and load. Moreover, attention should be given to younger patients who have had ART for longer periods. [ABSTRACT FROM AUTHOR]