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Background Almost half of the world’s population is at risk of malaria. Malaria is a parasitic vector borne disease spread by infected female Anopheles mosquitos. The protozoan parasite belongs to the genus Plasmodium group. In humans five main species are described, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. The recent years the WHO has reported that over 200 million malaria cases, and 400 000 deaths occur every year. Microscopy and rapid diagnostic tests (RDTs) are used in routine malaria diagnostics, but these methods have their challenges in accurate sensitivity and specificity. Aim To develop a new robust and user-friendly PCR method, highly sensitive and specific, and to assess and apply this method on relevant clinical material. Methods We developed a new single-step genus-specific conventional PCR targeting the cytochrome b gene (cytb) on the mitochondrial genome, and modified a species-specific conventional PCR targeting the chromosomal small subunit ribosomal RNA 18S locus. The methods were assessed and compared with routine microscopy and a gold standard nested 18S PCR using a Norwegian clinical collection (N=135), before applied in a multi-centre hospital-based study in India (N=1412). We also converted the conventional genus-specific cytb PCR to two real-time PCR assays applying SYBR Green and a TaqMan probe. The two assays were assessed using reference material, the Norwegian material (N=113) and a Tanzanian P. falciparum field collection (N= 74), and compared to five other relevant real-time PCR methods. Sequencing of the genus-specific cytb PCR products was also performed. Results Among the Norwegian sample collection (N=135) the novel genus-specific cytb PCR had a 100% sensitivity (28/28), nested 18S PCR 96% (27/28), and microscopy 93% (26/28). The 28 positive cytb PCR products were sequenced revealing six single nucleotide polymorphisms (SNPs) and one insert/deletion allowing for species determination of the 28 sequences. In the Indian multi-centre study, the cytb PCR found a malaria prevalence of 19% (268/1412). Overall, 46% P. falciparum and 38% P. vivax single infections were detected, while P. falciparum and P. vivax double infection was found in 11%, and P. malariae in 5% of the admitted fever patients. Routine microscopy and RDT had a low sensitivity compared to PCR, 29% and 24% respectively. Furthermore, the conventional cytb PCR was successfully converted to real-time PCR. The SYBR assay showed a higher sensitivity and specificity than using a probe. Focusing on detecting low-level parasitaemia, SYBR was also more beneficial to apply than probe due to melting curve analysis (MCA) can reveal unspecific binding or primer-dimers. Compared to the five other relevant real-time PCR methods our cytb SYBR PCR was the most sensitive genus-specific method. Conclusion Choice of amplification target is relevant for achieving high sensitivity in detecting low-level parasitaemia, and it is advantageous to use an intercalating fluorescence dye suitable for MCA. The highly sensitive, specific and user-friendly cytb SYBR PCR can be a useful tool in epidemiological and surveillance research, as well as for clinical malaria diagnostics. The method is genus-specific, which is an advantage in large screening projects. Among ambiguous samples, and in settings where species-specific PCR fails to detect low-level parasitaemia, confirmation and species identification by sequencing of the genus-specific real-time PCR products can be used as a contingency.