BackgroundGabon is a malaria-threatened country with a stable and hyperendemic transmission ofPlasmodium falciparummonoinfection. Malaria drug resistance is widely spread in many endemic countries around the world, including Gabon. The molecular surveillance of drug resistance to antifolates and artemisinin-based combination therapy (ACT) is one of the strategies for combating malaria. AsPlasmodiumparasites continue to develop resistance to currently available anti-malarial drugs, this study evaluated the frequency of the polymorphisms and genetic diversity associated with this phenomenon among the parasites isolates in Gabon.MethodsTo assess the spread of resistant haplotypes among the malaria-infected population of Libreville, single nucleotide polymorphisms linked to sulfadoxine–pyrimethamine (SP) and artemisinin drugs resistance were screened forP. falciparum dihydrofolate reductase(Pfdhfr),P. falciparum dihydropteroate synthase(Pfdhps), andP. falciparum kelch 13-propeller domain(Pfk13) point mutations.ResultsThe analysis of 70 malaria-positive patient samples screened for polymorphism showed 92.65%(n= 63) mutants vs. 7.35% (n= 5) wild parasite population inPfdhfr, with high prevalence mutations at S108N(88.24%,n= 60), N51I(85.29%,n= 58), C59R(79.41%,n= 54); however, I164L(2.94%,n= 2) showed low frequency mutation. No wild haplotype existed forPfdhps, and there were no mutations at the K540E, A581G, and A613T/S positions. However, the mutation rate at A437G(93.38%,n= 62) was the highest, followed by S436A/F(15.38%,n= 10). A higher frequency of quadruple IRNI–SGKAA (69.84%) than quintuple IRNI–(A/F)GKAA (7.94%) mutations was observed in thePfdhfr–Pfdhpscombination. Furthermore, none of the mutations associated with ACT resistance, especially those commonly found in Africa, were observed inPfk13.ConclusionsHigh polymorphism frequencies ofPfdhfrandPfdhpsgenes were observed, with alternative alanine/phenylalanine mutation at S436A/F (7.69%,n= 5) for the first time. Similar to that of other areas of the country, the patterns of multiple polymorphisms were consistent with selection owing to drug pressure. Although there was no evidence of a medication failure haplotype in the studied population, ACT drug efficacy should be regularly monitored in Libreville, Gabon.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12936-023-04615-1.