Background: Although the United Nations program on HIV/AIDS 90-90-90-targets recommends achieving 90% of viral suppression for patients on first-line antiretroviral therapy by 2020, virological failure is still high and it remains a global public health problem. Therefore, assessing the incidence and predictors of virological failure among adult HIV patients on first-line ART in Amhara regional referral hospitals, Ethiopia is vital to design appropriate prevention strategies for treatment failure and preventing the unnecessary switching to second-line regimens. Method: An institution-based retrospective follow-up study was conducted on 490 adult HIV patients. The simple random sampling technique was used, and data were entered into Epi data Version 4.2.0.0 and was exported to Stata version 14 for analysis. The proportional hazard assumption was checked, and the Weibull regression was fitted. Cox-Snell residual was used to test the goodness of fit, and the appropriate model was selected by AIC/BIC. Finally, an adjusted hazard ratio with a 95% CI was computed, and variables with P-value < 0.05 in the multivariable analysis were taken as significant predictors of virological failure. Results: The overall incidence rate of virological failure was 4.9 events per 1000 person-month observations (95%CI: 3.86-6.38). Users of CPT (AHR = 0.55, 95%CI: 0.31-0.97), poor adherence (AHR = 5.46, 95%CI: 3.07-9.74), CD4 Count <=200 cells/mm 3 (AHR = 3.9, 95%CI: 1.07-13.9) and 201-350 cells/mm 3 (AHR 4.1, 95%CI: 1.12-15) respectively, and NVP based first line drug regimen (AHR = 3.53, 95%CI: 1.73-7.21) were significantly associated with virological failure. Conclusion: The incidence rate of virological failure was high. CPT, poor adherence, low baseline CD4 count and NVP based first-line drug regimen were independent risk factors associated with virological failure. Therefore, strengthening HIV care intervention and addressing these significant predictors is highly recommended in the study setting.