The aim of this study was to assess the prevalence of mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes among pregnant women using sulfadoxine-pyrimethamine (SP) as an intermittent preventive treatment (IPTp). A molecular epidemiological study of P. falciparum parasite resistance markers to SP was conducted from August 2010 to February 2012 in Mukono district in central Uganda. DNA was extracted from 413 P. falciparum-positive samples. Real-time PCR, followed by melting curve analysis, was used to characterize point mutations in the Pfdhfr and Pfdhps genes that are associated with SP resistance. The prevalence of the single-nucleotide mutations in Pfdhfr at codons 51I, 59R, and 108N and in Pfdhps at codons 437G and 540E was high (>98%), reaching 100% fixation after one dose of SP, while the prevalence of 581G was 3.3% at baseline, reaching 12.5% after one dose of SP. At baseline, the prevalence of Pfdhfr and Pfdhps quintuple mutations was 89%, whereas the sextuple mutations (including 581G) were not prevalent (3.9%), reaching 16.7% after one dose of SP. However, the numbers of infections at follow-up visits were small, and hence there was insufficient statistical power to test whether there was a true rise in the prevalence of this allele. The overall high frequency of Pfdhfr and Pfdhps quintuple mutations throughout pregnancy excluded further analyses of possible associations between certain haplotypes and the risk of lower birth weight and anemia. However, women infected with P. falciparum had 1.3-g/dl-lower hemoglobin levels (P = 0.001) and delivered babies with a 400-g-lower birth weight (P = 0.001) compared to nonparasitemic women. Despite this, 44 women who were P. falciparum positive at baseline became negative after one or two doses of SP (i.e., 50.5%), implying that SP-IPTp still has some efficacy. P. falciparum resistance markers to SP are high in this population, whereas P. falciparum infection was associated with poor birth outcomes.