Strain diversity may play a role in delaying development of protective immunity in endemic areas. We evaluated genetic diversity of Plasmodium falciparum infected children before being treated with Sulphadoxine Pyrimethamine (SP) and Coartem™ in Southeastern Tanzania. Allelic diversity of P. falciparum strains were determined in order to further assist in correct estimation of recrudescent and new infections. P. falciparum isolated from 300 children aged 1-59 months was used in the study, where nested PCR followed by Restriction Fragment Length Polymorphism (RFLP) of highly polymorphic Merozoite surface protein 2 (msp2) was employed to understand the genetic diversity of the parasites population. Frequency of msp2 gene alleles was calculated and further associated with multiplicity of infection of children under five years of age. A total of 71 and 83 different msp2 alleles were found in Rufiji and Ulanga districts respectively. Children infected with either FC27 or 3D7 allelic type in Rufiji were 42% single, 55.3% double and 2.7% triple, while in Ulanga, 36.7% single, 62% double and 1.3% triple infections. Mean numbers of multiplicity of infections (MOI) in Rufiji and Ulanga were 1.6 and 1.3, respectively. These findings show a high genetic diversity of P. falciparum strains in study areas and low MOI could reflect production of susceptible parasites that immune response can accommodate or can be cleared by the drugs. Furthermore, 3D7 allelic type of msp2 gene was more prevalent than FC27 in Ulanga district, indicating association between msp2 allelic type and disease severity, hence predict possible vaccine candidate in the future.