Plasmodium falciparum and P. malariae occur endemically in many parts of Africa. Observations from malariotherapy patients suggest that co-infection with P. malariae may increase P. falciparum gametocyte production. We determined P. falciparum gametocyte prevalence and density by quantitative nucleic acid sequence-based amplification (QT-NASBA) after antimalarial treatment of Kenyan children with either P. falciparum mono-infection or P. falciparum and P. malariae mixed infection. In addition, we analyzed the relationship between mixed species infections and microscopic P. falciparum gametocyte prevalence in three datasets from previously published studies. In Kenyan children, QT-NASBA gametocyte density was increased in mixed species infections (P_0.03). We also observed higher microscopic prevalences of P. falciparum gametocytes in mixed species infections in studies from Tanzania and Kenya (odds ratio_2.15, 95% confidence interval_0.99–4.65 and 2.39, 1.58–3.63) but not in a study from Nigeria. These data suggest that co-infection with P. malariae is correlated with increased P. falciparum gametocytemia.