The goal of the present study was to assess the evolution of the in vitro chloroquine resistance and also the prevalence of pfcrt T76 and pfmdr1 Y86 mutations in Pikine from 2000 while chloroquine (CQ) was the first-line treatment of malaria to 2009 when artemisinin-based combination therapies (ACTs) are in use. We genotyped pfcrt K76T and pfmdr1 N86Y polymorphisms by PCR-RFLP and assessed in vitro CQ susceptibility by double-site enzyme-linked pLDH immunodetection (DELI) assay in Plasmodium falciparum isolates collected in Pikine, Senegal. The proportions of the pfcrt T76 allele in the light of the three different treatment policies were 72.4 % before CQ withdrawal (2000 to 2003), 47.2% while amodiaquine plus Fansidar was the first-line treatment (2004 to 2005), and 59.5 % since the ACT use was implemented (2006 to 2009). The prevalence of pfcrt T76 decreased significantly after CQ was stopped [X (2) = 6.54, P = 0.01 (2000-2003 versus 2004-2005)] and then slightly since ACTs have been implemented [X(2) = 1.12, P = 0.28 (2000-2003 versus 2006-2009)]. There were no significant differences on the prevalence of pfmdr1 Y86 throughout the three treatment policies. The DELI assay was carried out episodically in 2000 (n = 36), 2001 (n = 47), and 2009 (n = 37). The mean IC(50)s of the isolates to CQ in 2000 versus 2009 and 2001 versus 2009 are significantly different (P < 0.05). The Fisher exact test found a significant association between the presence of the pfcrt T76 mutant allele and in vitro resistance in 2000/2001 (P = 0.023), while in 2009 there were no association between both variables (P = 0.274). Mutant pfcrt T76 and pfmdr1 Y86 alleles and in vitro CQ-resistant strains are still circulating in Pikine. The official discontinuation of CQ use is not completely followed by its total withdrawal from private drug sellers, and the molecule still exerts pressure on local P. falciparum populations.