The accurate diagnosis of malaria starts with clinical suspicion, confirmed by reliable laboratory results. A hospital-based study, described here, was carried out in a malaria mesoendemic area in eastern Sudan, where the inhabitants are semi-immune to malaria, and the fever threshold of parasitemia is not above the detection level of microscopy. Thus, we hypothesized that patients with symptoms highly suggestive of cerebral malaria (CM), but aparasitemic by microscopy, may have submicroscopic parasitemia. Patients in our malaria clinic were screened by microscopy, and 120 individuals were selected for the study, including febrile patients with and without microscopically detectable parasitemia, and apparently healthy individuals. In the two former groups there were patients with severe anemia and deep coma. Polymerase chain reaction (PCR) for parasite detection and ELISA tests for measuring serum antibody levels were carried out on all blood samples. A majority of the febrile patients who were parasite negative by microscopy showed the presence of a Plasmodium falciparum infection by PCR. The occurrence of P. falciparum infection with parasitemia below the detection level of microscopy was recognized more often in patients with CM symptoms than in those with severe malarial anemia (SMA), and in older rather than younger patients. Patients clinically suspected (CS) of having CM ((CS)CM) mostly were infected with a single clone, and a large proportion of them acquired antibodies (Abs) against merozoite surface protein (MSP) antigens (Ags). The therapeutic response to quinine treatment was comparable between patients with (CS)CM and CM. In conclusion, uniquely in this setting, CM can be associated with sub-patent parasitemia; thus, a diagnostic tool more sensitive than microscopy is needed.