Plasmodium knowlesi malaria in humans is widely distributed and potentially life-threatening. This study aimed to determine the geographic distribution of P. knowlesi in Malaysia and investigate four fatal cases. Using nested PCR, P. knowlesi DNA was detected in 266 (27.7%) of 960 samples from Sarawak, 41 (83.7%) of 49 from Sabah, and all 5 from Pahang. All four fatal cases were hyperparasitemic and developed severe hepatorenal dysfunction. P. knowlesi is often misdiagnosed as P. malariae, which is more benign. However, P. knowlesi replicates every 24 hours, leading to severe disease. The study found that P. knowlesi infections are widespread in Malaysian Borneo and Peninsular Malaysia, and may extend beyond the Kapit Division. The four fatal cases were all hyperparasitemic and presented with severe symptoms. The study highlights the need for accurate diagnosis and prompt treatment of P. knowlesi malaria, as it can be life-threatening. The results suggest that P. knowlesi is a significant cause of severe malaria in Malaysia and that misdiagnosis can lead to inappropriate management. The study recommends that patients with symptoms resembling P. malariae should be diagnosed as P. knowlesi, especially those who have traveled to Southeast Asia. Further research is needed to define the spectrum of disease in P. knowlesi malaria.Plasmodium knowlesi malaria in humans is widely distributed and potentially life-threatening. This study aimed to determine the geographic distribution of P. knowlesi in Malaysia and investigate four fatal cases. Using nested PCR, P. knowlesi DNA was detected in 266 (27.7%) of 960 samples from Sarawak, 41 (83.7%) of 49 from Sabah, and all 5 from Pahang. All four fatal cases were hyperparasitemic and developed severe hepatorenal dysfunction. P. knowlesi is often misdiagnosed as P. malariae, which is more benign. However, P. knowlesi replicates every 24 hours, leading to severe disease. The study found that P. knowlesi infections are widespread in Malaysian Borneo and Peninsular Malaysia, and may extend beyond the Kapit Division. The four fatal cases were all hyperparasitemic and presented with severe symptoms. The study highlights the need for accurate diagnosis and prompt treatment of P. knowlesi malaria, as it can be life-threatening. The results suggest that P. knowlesi is a significant cause of severe malaria in Malaysia and that misdiagnosis can lead to inappropriate management. The study recommends that patients with symptoms resembling P. malariae should be diagnosed as P. knowlesi, especially those who have traveled to Southeast Asia. Further research is needed to define the spectrum of disease in P. knowlesi malaria.