Objective To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria. Methods In this study the nested multiplex malaria PCR was redesigned, targeting the 18S rRNA gene, to identify the fifth human Plasmodium species, Plasmodium knowlesi, together with the other human Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives. Results The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms. The technique showed high sensitivity (100%) and specificity (96%) when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos III and a published real-time PCR malaria assay. Conclusions The technique designed is an economical, sensitive and specific alternative to current diagnosis methods. Furthermore, the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.