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Background Recent data indicate that non- Plasmodium falciparum species may be more prevalent than thought in sub-Saharan Africa. Although Plasmodium malariae , Plasmodium ovale spp. and Plasmodium vivax are less severe than P. falciparum , treatment and control are more challenging, and their geographic distributions are not well characterized. Methods We randomly selected 3284 of 12 845 samples collected from cross-sectional surveys in 100 health facilities across 10 regions of Mainland Tanzania and performed quantitative real-time PCR to determine presence and parasitemia of each malaria species. Results P. falciparum was most prevalent, but P. malariae and P. ovale were found in all but 1 region, with high levels (>5%) of P. ovale in 7 regions. The highest P. malariae positivity rate was 4.5% in Mara and 8 regions had positivity rates ≥1%. We only detected 3 P. vivax infections, all in Kilimanjaro. While most nonfalciparum malaria-positive samples were coinfected with P. falciparum , 23.6% (n = 13 of 55) of P. malariae and 14.7% (n = 24 of 163) of P. ovale spp. were monoinfections. Conclusions P. falciparum remains by far the largest threat, but our data indicate that malaria elimination efforts in Tanzania will require increased surveillance and improved understanding of the biology of nonfalciparum species. [ABSTRACT FROM AUTHOR]