Malaria is a significant public health challenge in Ghana, with Plasmodium falciparum accounting for 80-90 % of infections. This study aimed to determine whether different climatic conditions, demographics, transmission patterns, and control practices in northern and southern Ghana result in differences in the genetic diversity, population structure, and evolutionary dynamics of P. falciparum. A total of 354 clinical samples collected in 2018 and 2021 were analyzed using the amplicon sequencing approach, targeting the CSP, AMA1, SERA2, and TRAP genes. Parasite populations from both northern and southern Ghana exhibited high haplotype diversity (Hd = 0.93-0.98). More than 70 % of the patient samples contained multiclonal infections, and the multiplicity of infection was significantly higher in northern (2.91) than in southern Ghana (2.42). Despite such a difference, extensive haplotype sharing was observed. The haplotype network and phylogenetic analyses highlighted substantial genetic connectivity among the parasite populations, likely driven by multiple factors, including human movement and shared vectors. Predominant haplotypes were consistent across regions and years, reflecting a stable pool of core haplotypes, which was not significantly impacted by contemporary control measures. In conclusion, this study underscored the high genetic diversity and connectivity of P. falciparum populations in Ghana despite different transmission ecology and the implementation of divergent control measures.