Over 80% of all malaria deaths occur in children under 5 years of age. Malaria controlstrategies have been progressively shifted to specific populations and/or areas to maximize effectiveness. Malaria is a significant public health problem in Ghana. Seasonal Malaria Chemoprevention (SMC) using a combination of sulfadoxine-pyrimethamine and amodiaquine has been implemented since 2015 in northern Ghana where malaria transmission is intense and seasonal. In this study, we estimated the prevalence of asymptomatic P. falciparum carriers in three ecological zones of Ghana, and compared the sensitivity and specificity of different molecular methods in identifying asymptomatic infections. Moreover, we examined the frequency of mutations in pfcrt, pfmdr1, pfdhfr, and pfdhps that relate to the ongoing SMC. A total of 535 asymptomatic schoolchildren were screened by microscopy and PCR (18s rRNA and TARE- 2) methods. Among all samples, 28.6% were detected as positive by 18S nested PCR, whereas 19.6% were detected by microscopy. A high PCR-based asymptomatic prevalence was observed in the north (51%) compared to in the central (27.8%) and south (16.9%). The prevalence of pfdhfr-N51I/C59R/S108N/pfdhps-A437G quadruple mutant associated with sulfadoxine-pyrimethamine resistance was significantly higher in the north where SMC was implemented. Compared to 18S rRNA, TARE-2 serves as a more sensitive molecular marker for detecting submicroscopic asymptomatic infections in high and low transmission settings. These findings establish a baseline for monitoring P. falciparum prevalence and resistance in response to SMC over time. Ghana is also one of the three African countries where the world’s first malaria vaccine, RTS, S, was launched recently. The vaccine contains part of the central repeat region and the complete C-terminal of the circumsporozoite protein (CSP) iv gene of the 3D7 strain. Polymorphism in the PfCSP protein has been reported from several parts of the world. However, whether RTS, S-induced immunity is PfCSP allele-dependent and if selection favors non-3D7 strains are unclear. This study aims to examine the genetic polymorphism of the PfCSP genes in clinical P. falciparum cases and provide a baseline of parasite diversity prior to vaccine implementation in Ghana. A total of 212 clinical samples were collected from Seikwa located in the Brong Afrong region where the vaccine is currently being deployed. Preliminary data indicated a high rate of polyclonal infections, with some samples harboring up to 3 clones based on the allele frequency among mapped reads. Parasite clones detected within the same host were not genetically similar to one another. Instead, they were distributed in various subclades and closely related to clones identified from other hosts. It is yet to be investigated if the high PfCSP haplotype diversity and low resemblance to the 3D7 strain have an impact on the anti-CSP immune response and thereby the efficacy of RTS,S.