BackgroundPrevention and treatment of malaria during pregnancy is crucial in dealing with maternal mortality and adverse fetal outcomes. The World Health Organization recommendation to treat all pregnant women with sulfadoxine-pyrimethamine (SP) through antenatal care structures was implemented in Kenya in the year 1998, but concerns about its effectiveness in preventing malaria in pregnancy has arisen due to the spread of SP resistant parasites. This study aimed to determine the prevalence of SP resistance markers in Plasmodium falciparum parasites isolated from pregnant women seeking antenatal care at Msambweni County Referral Hospital, located in coastal Kenya, between the year 2013 and 2015.MethodsThis hospital-based study included 106 malaria positive whole blood samples for analysis of SP resistance markers within the Pfdhfr gene (codons 51, 59 and 108) and Pfdhps gene (codons 437 and 540). The venous blood collected from all pregnant women was tested for malaria via light microscopy, then the malaria positive samples were separated into plasma and red cells and stored in a − 86° freezer for further studies. Archived red blood cells were processed for molecular characterization of SP resistance markers within the Pfdhfr and Pfdhps genes using real time PCR platform and Sanger sequencing.ResultsAll samples had at least one mutation in the genes associated with drug resistance; polymorphism prevalence of Pfdhfr51I, 59R and 108N was at 88.7%, 78.3% and 93.4%, respectively, while Pfdhps polymorphism accounted for 94.3% and 91.5% at 437G and 540E, respectively. Quintuple mutations (at all the five codons) conferring total SP resistance had the highest prevalence of 85.8%. Quadruple mutations were observed at a frequency of 10.4%, and 24.5% had a mixed outcome of both wildtype and mutant genotypes in the genes of interest.ConclusionThe data suggest a high prevalence of P. falciparum genetic variations conferring resistance to SP among pregnant women, which may explain reduced efficacy of IPTp treatment in Kenya. There is need for extensive SP resistance profiling in Kenya to inform IPTp drug choices for successful malaria prevention during pregnancy.