Through the regional control programme, Malaysia has been successfully reducing the incidence of Plasmodium falciparum and Plasmodium vivax infections. However, the incidence of zoonotic malaria Plasmodium knowlesi infection is increasing and now has been the major cause of malaria in Malaysia especially Malaysian Borneo. The emergence of knowlesi infection has threatened the malaria elimination programme which the government aims to reduce the overall malaria infections by 2020. Unlike other benign human Plasmodium spp., P. knowlesi can cause fatal infections. The aim of this study was to determine the incidence and distribution of five human malaria parasites including P. knowlesi in Peninsular Malaysia and Malaysian Borneo. A total of 112 blood samples were collected from seven states and district hospitals in Peninsular Malaysia and Malaysian Borneo from year 2015 to 2016. The samples were examined by microscopy and further confirmed by nested PCR assay targeting 18S rRNA gene of Plasmodium spp. Following the nested PCR assays, a total of 54 (48.2%) samples were positive for P. knowlesi infections, 12 (10.7%) cases were positive for P. vivax infections, followed by 7 (6.3%) cases of P. falciparum and 4 (3.5%) cases of P. malariae. There were 3 cases (2.7%) of mixed infections (P. knowlesi/P. vivax). However, no cases were identified as P. ovale. A total of 32 (28.6%) cases were found as negative infections. LoopMediated Isothermal Amplification Assay (LAMP) was performed to confirm inconclusive results produced by microscopy and nested PCR. P. knowlesi showed the highest prevalence in Sarawak (n= 30), Sabah (n=13), Pulau Pinang (n=5) and Pahang (n=6). PCR and LAMP was not able to detect a large number of microscopy positive samples due to DNA degradation during storage and shipping. Among all the states involved in this study, the highest prevalence of P. knowlesi infection was found in Sabah and Sarawak.