BackgroundMaternal knowledge on mother-to-child transmission (MTCT) and its prevention has been identified to enhance maternal testing and adherence to antiretroviral therapy (ART) regimen. Examining prevalence and associated factors on MTCT and its prevention among women provides empirical evidence for design and implementation of health strategies aimed at increasing MTCT knowledge and its elimination. This study therefore examined women’s comprehensive knowledge and associated factors on MTCT and its prevention among childbearing women in Rwanda.MethodsAnalysis was conducted on a weighted sample of 14,634 women from the 2020 Rwanda Demographic and Health Survey (RDHS). Dataset cleaning and missing value analysis was conducted. Chi square, bivariate and multivariable regression was then conducted in complex samples in SPSS. Alpha level set at p < 0.05 and at 95% Confidence Interval (95% CI). All analysis were adjusted for unequal probability sampling using survey weights. Bivariate and multivariable results were reported with crude and adjusted odds ratios.ResultsThe mean age was 29.2 years, SD-9.1. Prevalence of HIV testing and comprehensive knowledge on MTCT and its prevention among women in Rwanda was 79.6% and 65.1% respectively. Findings from this study showed that married women have higher odds (aOR = 1.18, 95% CI = 1.04–1.35) of comprehensive knowledge on MTCT and its prevention compared to those unmarried. Women who were living in southern (aOR = 1.23, 95%CI = 1.02–1.48) and eastern (aOR = 1.37, 95% CI = 1.13–1.66) parts of Rwanda were more likely to have adequate knowledge on MTCT of HIV and its prevention than those in Kigali. Also, women who received post-test counselling (aOR = 1.26, 95% CI = 1.01–2.11) have increased knowledge on MTCT than those who did not. Women with access to radio (aOR = 1.18, 95% CI = 1.06–1.32) and television (aOR = 1.25, 95% CI = 1.07–1.45) at least once a week were more likely to have adequate knowledge on MTCT and its prevention compared to those who do not in Rwanda.ConclusionThere is inadequate knowledge on MTCT and its elimination among women of reproductive age in Rwanda. Strategies to enhance knowledge on MTCT and its prevention among childbearing women should be adopted through rigorous educational sensitization campaigns using local media such as radio and television. Health services that focus on prevention of MTCT must emphasize post-test counselling.