Malaria remains a major public health concern in sub-Saharan Africa, and is emerging in regions with predominantly Duffy-negative populations. This study investigated prevalence and genetic diversity across three ecological zones in Cameroon. was detected in ~11% of febrile patients and 5.5% of asymptomatic individuals, all are Duffy-negatives. Clinical infections exhibited higher parasitemia than infections detected in the communities. Standard PvLDH-based RDTs produced false-negative results, even at high parasitemia levels, whereas molecular tools like qPCR demonstrated superior sensitivity. Genetic analysis of identified two prevalent mutations, I379L (73%) and E225K (61%), among samples, suggesting adaptive evolution. Phylogenetic analysis clustered Cameroonian isolates with those from Botswana but distinct from the Ethiopian and Sudanese isolates, indicating historical migration and local adaptation. The detection of asymptomatic cases highlights the potential for transmission, reinforcing the need for enhanced surveillance in both community and clinical settings.