PCR assay using designed primers was evaluated in detecting human malaria infection from whole blood and/or on Whatman filter-paper compared to conventional microscopy. Two DNA extraction methods were used for dried blood-spots; QIAamp mini kit and methanol-fixation/heat-extraction. A total of 118 cases were collected from 4 hospitals at Jazan district. Microscopic examination showed positivity in 66/118 samples (56%). Thin films showed parasitaemia from 1+ to 4+. In PCR assay, 79 samples (70%) were positive for the genus Plasmodium given 153 base pair PCR product. All microscopy positive samples were PCR positive but PCR detected 13 cases missed by microscopy. As for filter-paper spot samples, 68 samples (57.6%) were PCR positive when DNA was extracted by QIAamp mini kit whereas 49 (41.5%) by methanol-fixation/heat-extracion method. PCR sensitivity decreased by using DNA extracted from filter-paper compared to microscopy and whole blood PCR. But the DNA isolated from filter paper detected parasites in many microscopy negative samples.