BACKGROUND: Extensive genetic diversity in the Plasmodium falciparum circumsporozoite protein (PfCSP) is a major contributing factor to the moderate efficacy of the RTS,S/AS01 vaccine. The transmission intensity and rates of recombination within and between populations influence the extent of its genetic diversity. Understanding the extent and dynamics of PfCSP genetic diversity in different transmission settings will help to interpret the results of current RTS,S efficacy and Phase IV implementation trials conducted within and between populations in malaria-endemic areas such as Ghana. METHODS: Pfcsp sequences were retrieved from the Illumina-generated paired-end short-read sequences of 101 and 131 malaria samples from children aged 6-59 months presenting with clinical malaria at health facilities in Cape Coast (in the coastal belt) and Navrongo (Guinea savannah region), respectively, in Ghana. The sequences were mapped onto the 3D7 reference strain genome to yield high-quality genome-wide coding sequence data. Following data filtering and quality checks to remove missing data, 220 sequences were retained and analysed for the allele frequency spectrum, genetic diversity both within the host and between populations and signatures of selection. Population genetics tools were used to determine the extent and dynamics of Pfcsp diversity in P. falciparum from the two geographically distinct locations in Ghana. RESULTS: Pfcsp showed extensive diversity at the two sites, with the higher transmission site, Navrongo, exhibiting higher within-host and population-level diversity. The vaccine strain C-terminal epitope of Pfcsp was found in only 5.9% and 45.7% of the Navrongo and Cape Coast sequences, respectively. Between 1 and 6 amino acid variations were observed in the TH2R and TH3R epitope regions of PfCSP. Tajima's D was negatively skewed, especially for the population from Cape Coast, given the expected historical population expansion. In contrast, a positive Tajima's D was observed for the Navrongo P. falciparum population, consistent with balancing selection acting on the immuno-dominant TH2R and TH3R vaccine epitopes. CONCLUSION: The low frequencies of the Pfcsp vaccine haplotype in the analysed populations indicate a need for additional molecular and immuno-epidemiological studies with broader temporal and geographic sampling in endemic populations targeted for RTS,S application. These results have implications for the efficacy of the vaccine in Ghana and will inform the choice of alleles to be included in future multivalent or chimeric vaccines.