To assess adoption of World Health Organization (WHO) guidance into national policies for prevention of mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV) and to monitor implementation of guidelines at facility level in rural Malawi, South Africa and the United Republic of Tanzania. We summarized national PMTCT policies and WHO guidance for 15 indicators across the cascades of maternal and infant care over 2013-2016. Two survey rounds were conducted (2013-2015 and 2015-2016) in 46 health facilities serving five health and demographic surveillance system populations. We administered structured questionnaires to facility managers to describe service delivery. We report the proportions of facilities implementing each indicator and the frequency and durations of stock-outs of supplies, by site and survey round. In all countries, national policies influencing the maternal and infant PMTCT cascade of care aligned with WHO guidelines by 2016; most inter-country policy variations concerned linkage to routine HIV care. The proportion of facilities delivering post-test counselling, same-day antiretroviral therapy (ART) initiation, antenatal care and ART provision in the same building, and Option B+ increased or remained at 100% in all sites. Progress in implementing policies on infant diagnosis and treatment varied across sites. Stock-outs of HIV test kits or antiretroviral drugs in the past year declined overall, but were reported by at least one facility per site in both rounds. Progress has been made in implementing PMTCT policy in these settings. However, persistent gaps across the infant cascade of care and supply-chain challenges, risk undermining infant HIV elimination goals.