As a result of widespread antimalarial drug resistance, all African countries with endemic malaria have, in recent years, changed their malaria treatment policy. In Senegal, the health authorities changed from chloroquine (CQ) to a combination of sulfadoxine-pyrimethamine (SP) plus amodiaquine (AQ) in 2003. Since 2006, the artemisinin combination therapies (ACTs) artemether-lumefantrine (AL) and artesunate plus amodiaquine (AS/AQ) were adopted for uncomplicated malaria treatment. After several years of CQ withdrawal, the current study wished to determine the level of CQ resistance at the molecular level in selected sites in Senegal, because the scientific community is interested in using CQ again. Finger prick blood samples were collected from Plasmodium falciparum-positive children below the age of 10 years (N = 474) during cross-sectional surveys conducted in two study sites in Senegal with different malaria transmission levels. One site is in central Senegal, and the other site is in the southern part of the country. All samples were analyzed for single nucleotide polymorphisms (SNPs) in the P. falciparum CQ resistance transporter gene (Pfcrt; codons 72-76) using polymerase chain reaction (PCR) sequence-specific oligonucleotide probe (SSOP) enzyme-linked immunosorbent assay (ELISA) and real-time PCR methods. In total, the 72- to 76-codon region of Pfcrt was amplified in 449 blood samples (94.7%; 285 and 164 samples from the central and southern sites of Senegal, respectively). In both study areas, the prevalence of the Pfcrt wild-type single CVMNK haplotype was very high; in central Senegal, the prevalence was 70.5% in 2009 and 74.8% in 2010, and in southern Senegal, the prevalence was 65.4% in 2010 and 71.0% in 2011. Comparing data with older studies in Senegal, a sharp decline in the mutant type Pfcrt prevalence is evident: from 65%, 64%, and 59.5% in samples collected from various sites in 2000, 2001, and 2004 to approximately 30% in our study. A similar decrease in mutant type prevalence is noted in other neighboring countries. With the continued development of increased CQ susceptibility in many African countries, it may be possible to reintroduce CQ in the near future in a drug combination; it could possibly be given to non-vulnerable groups, but it demands close monitoring of possible reemergence of CQ resistance development.