A polymerase chain reaction (PCR) assay was developed for the specific detection of Plasmodium ovale, one of the four malaria parasites that infect humans. On the basis of sequence variation of the Plasmodium 18S ribosomal RNA (rRNA) gene, oligonucleotide primers for PCR were designed to amplify various fragments of the P. ovale gene. Using a recombinant plasmid with the complete P. ovale 18S rRNA gene as target, 59 primer combinations were tested so that at least one of the pairs was species-specific while the other primer was either genus conserved or P. ovale species-specific. Three primer pairs yielding DNA fragments at stringent conditions were further tested against genomic DNA of four human malaria species. This approach yielded P. ovale species-specific primer pairs that may be useful for further field testing.