Background: Drug resistance contributes to the global malaria burden. Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) polymorphisms confer resistance to sulphadoxine-pyrimethamine (SP). Methods: The study assessed the frequency of SP resistance-conferring polymorphisms in Plasmodium falciparum-positive samples from two clinical studies in Lambarene. Their role on treatment responses and transmission potential was studied in an efficacy open-label clinical trial with a 28-day follow-up in 29 children under five with uncomplicated malaria. Results: SP was well tolerated by all subjects in vivo. Three subjects were excluded from per-protocol analysis. PCR-corrected, 12/26 (46%) achieved an adequate clinical and parasitological response, 13/26 (50%) were late parasitological failures, while 1/26 (4%) had an early treatment failure, resulting in early trial discontinuation. Of 106 isolates, 98 (92%) carried the triple mutant dhfr haplotype. Three point mutations were found in dhps in a variety of haplotypic configurations. The 437G + 540E double mutant allele was found for the first time in Gabon. Conclusions: There is a high prevalence of dhfr triple mutant with some dhps point mutations in Gabon, in line with treatment failures observed, and molecular markers of SP resistance should be closely monitored.