Malaria Transmission Blocking Vaccine (MTBV) candidates capable of eliciting effective and long-lasting protection, thereby blocking or reducing community Plasmodium falciparum transmission rates are becoming more fashionable. Domain 1 of pre-fertilization P. falciparum surface protein 230 (Pfs230 D1), Pfs48/45 D3, and post-fertility protein Pfs25 are key fronted vaccine candidates. Aside from eliciting a strong antibody response, their candidacy's success will be determined by their ability to overcome obstacles such as antigenic variations that may affect their functionality, resulting in strain transcendence or allele-specific immunity, or loss of antigenicity due to conformation of antibody binding epitopes or antigen structure. This study determined polymorphisms with functional significance on the immunodominant domains of the three antigens in P. falciparum populations from different rural parts of western Kenya with varying transmission intensities. Clinical blood smears, dry blood spots and patients’ demographic data were collected at health facilities in Malaria-endemic Homa Bay (endemic area with ongoing indoor residual spray), Kisumu (endemic area without IRS), and Kisii (epidemic prone highland). Microscopy and real-time polymerase chain reaction were used to detect microscopic and submicroscopic infections, respectively. On parasite positive DNA samples, each target gene was genotyped and genetic computations were performed. In epidemic-prone rural area of Kisii highland, there were 231/1115 (20.7%) positive malaria cases. Morara and Nyabikondo village had the most microscopic infections (24/57, 42%). Submicroscopic infections were uncommon (14.7%) and more common in females (19.7%) and adults (21.1%). In malaria endemic zone of Homa Bay, clinical microscopic infections were low (14.7%). The majority of positive clinical cases (36%, 112/313) were submicroscopic, resulting in an overall prevalence of 45.2%. Submicroscopic infections were more prevalent in females (35.6%, 72/202) than in males (24.2%, 40/165). Among 156 iiigenotyped samples, the Pfs230 D1 had low nucleotide diversity (π=0.15×10-2), with variation observed per study site. Six segregating sites (1616, 1813, 1955, 1964, 1967, and 1983) were identified, each resulting in nonsynonymous mutations (I539T, G605S, T652R, E655V, T656N, and K661N) and eight haplotypes. Recombination, inbreeding, purifying and balancing selection were key drivers of these mutations. The Pfs48/45 D3 sequences of the 118 clinical isolates had a high conservation index and low nucleotide diversity (0.063×10-2). Three polymorphic sites were identified: 911, 940, and 979, which resulted in nonsynonymous mutations (V304D, L314I, and C327G). C327G polymorphism occurred on one of the cysteine residues involved in structural conformation. Except for recombination, the drivers for these mutations were similar to those for Pfs230 D1. On the 177 Pfs25 sequences, low mutation frequencies (1.3% to 7.7%) were observed, resulting in a low nucleotide diversity (0.04×10-2). Ten dimorphic codons were discovered (H41H, L42M, I83I, C110C, C115W, L122L, T124T, T130T, V132I, and V143G), nine of which were novel (H41H, L42M, I83I, C110C, C115W, L122L, T124T, T130T, V132I, and V132I). Two polymorphic codons (C110C and C115W) were of functional importance and occurred on cysteine residues. There was strong purifying selection at all polymorphic loci, as well as a high level of inbreeding. This study established that there are a high number of clinical malaria cases in epidemic-prone zone of Kisii highland, and two villages are potential sentinel sites for future public health intervention if a malaria epidemic occurs. Despite low levels of microscopic infection in malaria endemic zone (Homa Bay), a high proportion of positive (submicroscopic) malaria cases are missed in health facilities; a more sensitive diagnosis test should be used to supplement microscopy. This will help to reduce the fraction of false negative results, potentially lowering the risk of escalating or maintaining residual P. falciparum community transmissions. With the presence of polymorphic sites of functional significance in Pfs230 D1, Pfs48/45 D3, and Pfs25, there is a need for further immunological investigation of the effect ivof these polymorphisms when designing TBVs based on the sequences of these genes and their potential impact on antibody binding epitopes

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