The efficacy of Sulphadoxine/Pyrimethamine (SP) in Plasmodium falciparum malaria treatment was increasingly compromised by development of parasites' resistance. Saudi Arabia shifted to new combinations including Artesunat compound during the last decade. We investigated the occurrence of mutations in P. falciparum dihydrofolate reductase
(Pfdhfr) N51I, C59R and S108N and P. falciparum dihyropteroate synthetase (Pfdhps) A437G and K540D as major indicators of SP resistance in stored DNA extracts of 41 P. falciparum infected specimens collected from KSA southern endemic regions between 2012 and 2014. Analysis of alleles' polymorphisms by Nested-PCR-RFLP showed that 68%, 7%, and 24% of samples carried parasites with Pfdhfr 51I, 59R, and 108N mutant type alleles, respectively. Only one isolate's genotype shared both mutations 51I and 108N. All parasites conserved wild type alleles at codons 437 and 540 of Pfdhps gene.