In Nigeria, the discontinued use of chloroquine for malaria treatment was officially announced in 2005. A few available reports have shown a persistent high prevalence of the major biomarker of chloroquine resistance in southwest Nigeria. However, information on its prevalence in rural and urban areas is scanty. We investigated possible factors associated with the prevalence of a biomarker for chloroquine-resistance in Ogun State, southwest Nigeria. Parasite DNA was extracted from dried blood spots collected by finger-prick in malaria symptomatic and asymptomatic subjects attending the urban-based State General Hospital and a rural-based Primary Health Centre. A structured questionnaire was used to collect data on malaria/fever treatment history. Nested Polymerase Chain Reaction (PCR) followed by Restriction Fragment Length Polymorphisms (RFLP) analysis was used to detect mutations in the P. falciparum chloroquine resistance transporter (Pfcrt). Of the 243 participants recruited for this study, 56 were found to harbour P. falciparum parasites, of which 62.5% (35/56) showed symptoms of malaria. Prevalence of P. falciparum chloroquine-resistant strains (Pfcrt K76T) was 69.6%. The prevalence of Pfcrt K76T recorded in the rural area (91.7%) was significantly higher (P<0.05) than that in the urban area (53.1%). There was no correlation between prevalence of chloroquine-resistant strains and malaria symptoms in the rural area. However, prevalence of chloroquine-resistant strains was significantly higher in malaria-symptomatic subjects from the urban area. Drug-resistant P. falciparum strains recorded in the rural area were associated with self-medication and patronage of drug vendors who continue to sell chloroquine. These findings present the importance of continuous surveillance of biomarkers indicating drug resistance especially now that antimalarial drug resistance is a threat to malaria eradication.