Four Plasmodium species cause malaria in humans. Most malaria-endemic regions feature mixed infections involving two or more of these species. Factors contributing to heterogeneous parasite species and disease distribution include differences in genetic polymorphisms underlying parasite drug resistance and host susceptibility, mosquito vector ecology and transmission seasonality. It is suggested that unknown factors limit mixed Plasmodium species infections, and that mixed-species infections protect against severe Plasmodium falciparum malaria. Careful examination of methods used to detect these parasites and interpretation of individual- and population-based data are necessary to understand the influence of mixed Plasmodium species infections on malarial disease. This should ensure that deployment of future antimalarial vaccines and drugs will be conducted in a safe and timely manner.