Sulfadoxine/pyrimethamine (SP) is considered an alternative treatment for acute uncomplicated malaria caused by Plasmodium falciparum resistant to chloroquine. However, the appearance of resistance to this drug has been reported since its initial use in Colombia. Molecular analysis of the dihydrofolate reductase gene indicates a correlation between in vitro resistance to SP and the Asn-108 point mutation. Little is known about the association of this point mutation and in vivo resistance to SP. We used a mutation-specific polymerase chain reaction strategy to analyze the presence of the Asn-108 point mutation in 48 clinical samples with adequate clinical response (ACR), 2 early treatment failures (ETF), and 1 late treatment failure (LTF). The Asn-108 mutation was detected in 36 of the ACR samples and in all of the ETF and LTF samples. Eleven ACR samples amplified with the wild-type-specific primer and one amplified with the primer for the Thr-108 mutation described for resistance to cycloguanil. These results suggest that the Asn-108 marker may not be useful in predicting SP treatment failure.