Data on prevalence of antimalarial molecular resistance markers in pregnant women in Ghana is scarce. Prevalence of single nucleotide polymorphisms/haplotypes in the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes was assessed in a cross-sectional study involving 200 pregnant women. Almost 90% of infections were wild type at the Pfcrt gene whereas the Pfmdr1 NFD mutant haplotype occurred in 43% of samples. Prevalence of Pfdhfr/Pfdhps quadruple mutation was 92.6% whereas Pfdfr/Pfdhps quintuple mutation with K540E was not observed. The study provides important updates of antimalarial resistance markers in Ghanaian pregnant women and suggests increased tolerance to one of the first-line treatment options in Ghana: artemether-lumefantrine. The data support the view that sulfadoxine-pyrimethamine is still efficacious for intermittent preventive treatment in Ghana, but the impact of increased doses on selection of mutations needs to be assessed. Continuing the surveillance of resistance markers is important to inform changes in antimalarial drug policy in pregnancy.