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Background: The return of chloroquine-sensitive Plasmodium falciparum in sub-Saharan Africa countries offers the opportunity for the reintroduction of chloroquine (CQ) either in combination with other drugs or as a single therapy for the management of malaria. This study assesses the influence of individual study sites on the selection of CQ sensitive P. falciparum markers in the Central region of Ghana. Methods: Genomic DNA was extracted from an archived filter paper blood blot from Cape Coast, Elmina, Assin Fosu, and Twifo Praso using the Chelex DNA extraction method. The age metadata of the patients from whom the blood spots were taken was collected. The prevalence of CQ-sensitive markers of pfcrt K76 and pfmdr1 N86 was performed using nested PCR and RFLP. The data were analysed using Chi-square and Odd ratio. Results: The overall prevalence of CQ-sensitive P. falciparum markers, pfcrt K76 and pfmdr1 N86 in the Central Region of Ghana were 142 out of 184 (77.17%) and 180 out of 184 (97.83%), respectively. The distribution of pfcrt K76 was assessed among the age groups per the individual study sites. 12 out of 33 (36.36%), 8 out of 33 (24.24%) and 6 out of 33 (18.18%) of pfcrt K76 CQ-sensitive marker were isolated from age 0 to 5 years, 16 to 30 years and 31 to 45 years old respectively at Cape Coast. Assin Fosu and Twifo Praso had the highest pfcrt K76 prevalence in 0–5 years, followed by 16–30 years and 6–15 years of age. The results showed that there was a significant prevalence of pfcrt K76 in all study sites; Cape Coast (χ2 = 26.48, p < 0.0001), Assin Fosu (χ2 = 37.67, p < 0.0001), Twifo Praso (χ2 = 32.25, p < 0.0001) and Elmina (χ2 = 17.88, p < 0.0001). Again, the likelihood to detect pfcrt K76 (OR (95% CI) was 7.105 (3.118–17.14), p < 0.0001 and pfmdr1 (2.028 (1.065–3.790), p < 0.001) among P. falciparum isolates from Cape Coast to be seven times and two times, respectively. Conclusion: The study showed a significant selection and expansion of chloroquine-sensitive P. falciparum markers in all the selected study areas in the Central region. This finding has a significant implication for the future treatment, management, and control of P. falciparum malaria. [ABSTRACT FROM AUTHOR]