Malaria infections in South Africa have surged to alarming levels over the last few years. This study was undertaken to establish the occurrence of antifolate-resistant parasites and of mixed-species malaria infections in the Mpumalanga province of South Africa. Blood samples from infected patients were subjected to molecular screening methods based on parasite nucleic acid properties. The assays consisted of restriction enzyme analyses specific for point mutations in the polymerase chain reaction (PCR) amplified gene of the antifolate target enzyme, dihydrofolate reductase (DHFR), which are known to confer drug resistance. Concurrently, the infective species were identified using PCR amplification of the small-subunit ribosomal RNA genes. Of the 56 samples analysed, six (12%) showed mixed-species infections, four being R falciparum-R ovale and two P. falciparum-P. vivax mixtures. Analyses of resistance to the antifolates revealed that out of 21 single R falciparum-infected samples tested, 43% contained parasites resistant to pyrimethamine treatment. The mutations were located in codon 108 and all were substitutions of Ser to Asn. Despite the relatively small sample size, these results suggest that malaria resistance to antifolates in the Mpumalanga province, which includes the Kruger National Park, could significantly influence the chemotherapeutic regimens employed for the prophylaxis and treatment of the disease.