The genetic make-up of malaria parasite is potent for understanding the parasite virulence, designing antimalarial vaccine and evaluating the impact of malaria control measures. There is a paucity of information on genetic structure of Plasmodium falciparum in Jharkhand, India where malaria is rampant and this study aimed to establish molecular characterization of P. falciparum field isolates from Jharkhand measured with two highly polymorphic genetic markers, i.e. the merozoite surface proteins (MSPs) 1 and 2. The genetic diversity of P. falciparum population from low transmission area, Ranchi, Bokaro and Hazaribagh and highly malarious area, Latehar and Palamau districts of Jharkhand were evaluated by polymerase chain reaction-sequencing analyzing msp-1 and msp-2 genes to explore the genetic structure of parasite from this understudied region. A total of 134 P. falciparum isolates were analyzed by polymorphic regions of msp-1 and msp-2 and classified according to prevalence of allelic families. The majority of patients from all the five sites had mean monoclonal infections of 67·1 and 60·4% of P. falciparum for msp-1 and msp-2, respectively, whereas, mean multiple genotypes of 32·8 and 39·5% for msp-1 and msp-2, respectively. Interestingly, we observed higher multiclonal infection in low transmission area as compared to highly malarious area in the case of msp-1 genotypes, whereas in msp-2 higher multiclonal infection was observed in highly malarious area compared to low transmission area. The overall multiplicities of infection of msp-1 and msp-2 were 1·38 and 1·39, respectively. This is the first report on molecular characterization of P. falciparum field isolates from Jharkhand. The genetic diversity and allelic distribution found in this study is somewhat similar to other reports from India and Southeast Asian countries. However, P. falciparum infection can be highly complex and diverse in these disease-endemic regions of Jharkhand, suggesting continual genetic mixing that could have significant implications for the use of antimalarial drugs and vaccines.