BACKGROUND: Plasmodium falciparum prevalence (PfPR) is a widely used metric for assessing malaria transmission intensity. This study was carried out concurrently with the RTS,S/AS01 candidate malaria vaccine Phase III trial and estimated PfPR over ≤ 4 standardized cross-sectional surveys. METHODS: This epidemiology study (NCT01190202) was conducted in 8 sites from 6 countries (Burkina Faso, Gabon, Ghana, Kenya, Malawi, and Tanzania), between March 2011 and December 2013. Participants were enrolled in a 2:1:1 ratio according to age category: 6 months-4 years, 5-19 years, and ≥ 20 years, respectively, per year and per centre. All sites carried out surveys 1-3 while survey 4 was conducted only in 3 sites. Surveys were usually performed during the peak malaria parasite transmission season, in one home visit, when medical history and malaria risk factors/prevention measures were collected, and a blood sample taken for rapid diagnostic test, microscopy, and haemoglobin measurement. PfPR was estimated by site and age category. RESULTS: Overall, 6401 (survey 1), 6411 (survey 2), 6400 (survey 3), and 2399 (survey 4) individuals were included in the analyses. In the 6 months-4 years age group, the lowest prevalence (assessed using microscopy) was observed in 2 Tanzanian centres (4.6% for Korogwe and 9.95% for Bagamoyo) and Lambaréné, Gabon (6.0%), while the highest PfPR was recorded for Nanoro, Burkina Faso (52.5%). PfPR significantly decreased over the 3 years in Agogo (Ghana), Kombewa (Kenya), Lilongwe (Malawi), and Bagamoyo (Tanzania), and a trend for increased PfPR was observed over the 4 surveys for Kintampo, Ghana. Over the 4 surveys, for all sites, PfPR was predominantly higher in the 5-19 years group than in the other age categories. Occurrence of fever and anaemia was associated with high P. falciparum parasitaemia. Univariate analyses showed a significant association of anti-malarial treatment in 4 surveys (odds ratios [ORs]: 0.52, 0.52, 0.68, 0.41) and bed net use in 2 surveys (ORs: 0.63, 0.68, 1.03, 1.78) with lower risk of malaria infection. CONCLUSION: Local PfPR differed substantially between sites and age groups. In children 6 months-4 years old, a significant decrease in prevalence over the 3 years was observed in 4 out of the 8 study sites. Trial registration Clinical Trials.gov identifier: NCT01190202:NCT. GSK Study ID numbers: 114001.