Background Human immunodeficiency virus (HIV) infected children represent a very vulnerable population for anti-retroviral therapy (ART) drug resistance. As a global target, 90% of patients receiving ART should have HIV-RNA viral suppression. A threshold of > 1000 RNA copies/ml is used to define non-suppressed viral load. If it is confirmed in the laboratory, adherence should be addressed and should be followed by the switch to second-line ART. Therefore, the aim of this study was to assess the rate of viral load suppression among children tested at the Amhara Public Health Institute (APHI), Bahir Dar. Methods Institutional based cross-sectional study design was conducted from July 01, 2017 to June 30, 2018, in children under the age of 15 years. Socio-demographic, clinical and HIV1RNA viral load data were collected from the excel database. The data were analyzed in SPSS 20.0 statistical software. Results A total of 1567 children, age ranged from one to 14 years, were tested for HIV viral load. Of which, about 54% were males. Children were treated using nevirapine-based (76.7%), efavirenz-based (21.8%) and protease inhibitor-based (1.5%) anti-retroviral drugs. Non-suppressed HIV viral load was found in 28.3% of the participants. High viral load (> 1000 cp/ml) were found in 24% of the children below the age of five years. Children on nevirapine-based treatment had about two times more non-suppressed viral load (Adjusted odds ratio [AOR]: 1.90; 95%CI: 1.41–2.56; P < 0.001) compared to those who had efavirenz-based treatment. However, adherence (P: 0.204) was not associated with non-suppressed viral load. Conclusions There was a high rate of non-suppressed HIV viral load among children tested at APHI. Specifically, the odds of having a non-suppressed viral load was higher in NVP based treatment users. Hence, comprehensive management and follow up of children on ART, and testing for resistance as well as viral load could help to reduce the problem in advance.