Background Assessment of the genetic diversity of Plasmodium falciparum parasites from various malaria transmission settings could help to define tailored local strategies for malaria control and elimination. Such assessments are currently scarce in Madagascar. The study presented here aimed to bridge this gap by investigating the genetic diversity of P. falciparum populations in three epidemiological strata (Equatorial, Tropical and Fringes) in Madagascar. Methods Two-hundred and sixty-six P. falciparum isolates were obtained from patients with uncomplicated malaria enrolled in clinical drug efficacy studies conducted at health centres in Tsaratanana (Equatorial stratum), Antanimbary (Tropical stratum) and Anjoma Ramartina (Fringes) in 2013 and 2016. Parasite DNA was extracted from blood samples collected before anti-malarial treatment. Plasmodium species were identified by nested PCR targeting the 18 S rRNA gene. The genetic profiles of P. falciparum parasites were defined by allele-specific nested PCR on the polymorphic regions of the msp - 1 and msp - 2 genes. Results Fifty-eight alleles were detected in the P. falciparum samples tested: 18 alleles for msp-1 and 40 for msp-2 . K1 (62.9%, 139/221) and FC27 (69.5%, 114/164) were the principal msp-1 and msp-2 allele families detected, although the proportions of the msp-1 and msp-2 alleles varied significantly between sites. Polyclonal infections were more frequent at sites in the Equatorial stratum (69.8%) than at sites in the Tropical stratum (60.5%) or Fringes (58.1%). Population genetics analyses showed that genetic diversity was similar between sites and that parasite flow within sites was limited. Conclusions This study provides recent information about the genetic diversity of P. falciparum populations in three transmission strata in Madagascar, and valuable baseline data for further evaluation of the impact of the control measures implemented in Madagascar.