BackgroundPediatric HIV infection is mainly caused by Mother-to-child transmission (MTCT). Without any effective medical intervention, 15-45% of infants born to HIV-positive women will become infected with HIV. The contribution of viral non-suppression for MTCT is high. Hence, this study was to determine the magnitude of HIV viral non-suppression, and associated factors among PMTCT Mothers in Ethiopia.MethodsThe study was conducted from April 1, 2023, to December 31/ 2024 at 16 public health facilities. An institutional-based cross-sectional study was employed and 496 HIV-positive pregnant and lactating women on ART were included. Data was collected using a prechecked questionnaire with paper mode and ODK (Open Data Kit) for each participant. Venous blood was collected using two Ethylenediamine Tetra acetic Acid (EDTA) tubes for HIV viral load, CD4 and Hgb test. HIV viral load tests were conducted at the EPHI (Ethiopian Public Health Institute) National HIV reference laboratory using the COBAS 4800 System (Roche Molecular Diagnostics). Data was edited and exported to SPSS from the ODK data file, and finally data analysis was performed using SPSS version 26 software.Results496 PMTCT Mothers were included in this study, the prevalence of HIV viral non-suppression was 2.2% (95% CI; 1.1-3.9), the rate of viral detectability was 21.8 % (95% CI; 18.2-25.7) and the rate of low-level viremia (LLV) was 9.1%. Family size (X2=7.20; p<0.001), a poor and fair level of Adherence (X2=18.553; p<0.001), exposure to opportunistic infection (X2=25.29; p<0.001), survey time WHO clinical stages II & III (X2=25.29; p<0.001), HIV status non-disclosure other than Health care workers (HCWs) (X2=4.408; p=0.036), and low survey time CD4 count (<350 cells/ul) ( X2= 15.989; p<0.001) were significantly associated to the rate of HIV viral non-suppression.ConclusionsIn this study, HIV viral non-suppression among pregnant and lactating women is relatively low (meet the UNAIDS 2030 target). However, Family size, level of Adherence, exposure to opportunistic infection, WHO clinical stage level II and III, HIV status disclosure and low CD4 count were significantly associated with the prevalence of viral non-suppression. To achieve persistent HIV viral suppression enhanced adherence and counselling services should be provided, tailored to the needs of the specific target group.