Several studies have shown the efficacy of the intermittent preventive treatment (IPT) using sulfadoxine-pyrimethamine (SP) coupled with the expanded program of immunization (EPI) in infants. However, its adoption as a strategy is conditioned by the long-term efficacy of SP. The impact of IPT-SP coupled with the EPI on the prevalence of markers of resistance to SP was evaluated during this study conducted in Southern Senegal. Three cross-sectional surveys in two health districts (IPT+) were conducted prior to the implementation, 1 year, and 2 years after. A third district located between the two districts served as a test zone (IPT-). PCR tests were carried out from filter papers collected in children under five for the two first measures and from positive rapid diagnostic tests in the same population for the third measure. Mutations in codons 51, 59, and 108 of the DHFR gene and in codons 437 and 540 of the DHPS were analyzed. The results showed that the prevalence of DHFR triple mutation was more frequent after 2 years in IPT+ areas. Regarding quadruple mutation, DHFR (51, 59, and 108) and DHPS (437), no difference was noted between the two areas. The quintuple mutation was not observed after 2 years of implementation in both areas. However, an individual analysis showed significant differences in the individual mutation points 51, 59, 108, and 437. This study reveals that despite an increase in the prevalence of individual mutations, the IPT-SP coupled with the EPI has no major impact on DHFR and DHPS combined mutations.