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<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>and <it>Plasmodium falciparum </it>are the major causative agents of malaria. While knowledge of the genetic structure of malaria parasites is useful for understanding the evolution of parasite virulence, designing anti-malarial vaccines and assessing the impact of malaria control measures, there is a paucity of information on genetic diversity of these two malaria parasites in Pakistan. This study sought to shed some light on the genetic structure of <it>P. vivax </it>and <it>P. falciparum </it>in this understudied region.</p> <p>Methods</p> <p>The genetic diversities of <it>P. vivax </it>and <it>P. falciparum </it>populations from the densely populated, malaria-endemic Bannu district of Pakistan were evaluated by analysis of their merozoite surface protein (<it>msp</it>) genes by PCR-RFLP. Specifically, the <it>Pvmsp-3α </it>and <it>Pvmsp-3β </it>genes of <it>P. vivax </it>and the <it>Pfmsp-1 </it>and <it>Pfmsp-2 </it>genes of <it>P. falciparum </it>were analysed.</p> <p>Results</p> <p>In <it>P. vivax</it>, genotyping of <it>Pvmsp-3α </it>and <it>Pvmsp-3β </it>genes showed a high level of diversity at these loci. Four distinct allele groups: A (1.9 kb), B (1.5 kb), C (1.2 kb), and D (0.3 kb) were detected for <it>Pvmsp</it>-<it>3α</it>, type A being the most prevalent (82%). Conversely, amplification of the <it>P. vivax msp</it>-<it>3β </it>locus produced two allele groups: A (1.7-2.2 kb, 62%) and B (1.4-1.5 kb, 33%), with 5% mixed-strain infections. Restriction analysis of <it>Pvmsp-3α </it>and <it>Pvmsp-3β </it>yielded 12 and 8 distinct alleles, respectively, with a combined mixed genotype prevalence of 20%. In <it>P. falciparum</it>, all three known genotypes of <it>Pfmsp-1 </it>and two of <it>Pfmsp-2 </it>were observed, with MAD20 occurring in 67% and 3D7/IC in 65% of the isolates, respectively. Overall, 24% <it>P. falciparum </it>samples exhibited mixed-strain infections.</p> <p>Conclusion</p> <p>These results indicate that both <it>P. vivax </it>and <it>P. falciparum </it>populations in Pakistan are highly diverse.</p>