Polymorphisms in the genes encoding the merozoite surface proteins msp-1 and msp-2 are widely used markers for characterizing the genetic diversity of Plasmodium falciparum. This study aimed to compare the genetic diversity of circulating parasite strains in rural and urban settings in the Republic of Congo after the introduction of artemisinin-based combination therapy (ACT) in 2006. A cross-sectional survey was conducted from March to September 2021 in rural and urban areas close to Brazzaville, during whichPlasmodiuminfection was detected using microscopy (and nested-PCR for submicroscopic infection). The genes coding formerozoite proteins-1and-2were genotyped by allele-specific nested PCR. Totals of 397 (72.4%) and 151 (27.6%)P. falciparumisolates were collected in rural and urban areas, respectively. The K1/msp-1 and FC27/msp-2 allelic families were predominant both in rural (39% and 64%, respectively) and urban (45.4% and 54.5% respectively) areas. The multiplicity of infection (MOI) was higher (p= 0.0006) in rural areas (2.9) compared to urban settings (2.4). The rainy season and the positive microscopic infection were associated with an increase in MOI. These findings reveal a higherP. falciparumgenetic diversity and MOI in the rural setting of the Republic of Congo, which is influenced by the season and the participant clinical status.