Due to increasing trend in chloroquine resistance, the antifolate (sulpha-pyrimethamine combination) drugs are gaining more importance in the treatment of uncomplicated falciparum malaria. The efficacy of sulpha-pyrimethamine combinations in the treatment is compromised by the development of resistance in parasite. The occurrence of mutations at active sites in Plasmodium falciparum gene sequences coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) confer resistance to pyrimethamine and sulphadoxine. This study presents the characterization of a P. falciparum sample from a patient who did not respond to standard doses of a pyrimethamine/sulpha regimen. Although parasitaemia fell rapidly, the infection had not resolved six days later as because the response to treatment selected resistant sub-population. The in vitro drug sensitivity assays demonstrated resistance to pyrimethamine, sulphadoxine and cycloguanil; while polymerase chain reaction (PCR) and restriction digest based methods indicated that at known drug resistant loci the isolate had a genotype of DHFR Val-16 and Thr-108 previously only associated with cycloguanil resistance. As per the published reports this type of paired mutations in natural isolates are rare. It is of considerable interest to carry out studies on alleles on alleles of this gene in relation to resistance at epidemiological level.