Malaria is one of the important parasite transmitted diseases in Sabah, Malaysian Borneo that is covered by tropical rainforest. This six-months cross-sectional study aimed to accurately identify the human malaria parasites and to determine the misdiagnosis rate of human malaria parasites in the interior division of Sabah using nested PCR. A total of 243 blood spot samples from patients who had requested for Blood film for malaria parasites (BFMP) were collected from four study sites namely, Keningau, Tenom, Tambunan and Nabawan. There were 16.1% P.malariae, 10.3% P.vivax, 7% P.falciparum, 1.2% mixed infection and 65.4% negative cases based on microscopic examination. However, nested PCR indicated that P.knowlesi (58.9%), P.falciparum (18.7%), P.vivax (18.7%), mixed infection (3.7%) and no P.malariae infection were detected in these samples. Only 35% of 243 samoles gave consistent PCR and microscopic finsings. The highest malaria cases found in Keningau were P.falciparum (36.1%) whereas the prevalence of P.knowlesi (85.7%) was higher in Tambunan and Tenom (77.8%). This study reveals the actual transmission of different Plasmodium species in the interior region of Sabah.