In Ethiopia, chloroquine was the first line anti-malarial drug used for the treatment ofrnuncomplicated falciparum malaria for more than 40 years, until very high treatment failurernrates were demonstrated through studies conducted in 1997/98. Following these findings,rnthe national drug policy for uncomplicated falciparum malaria treatment was shifted tornsulfadoxine/pyrimethamine (SP). Unfortunately, SP resistance became widely prevalentrnsoon after its introduction and was replaced by artemether-lumefantrine (AL) in 2004. Inrnorder to assess the impact of this policy change on the prevalence of molecular markersrnlinked to chloroquine resistance in Harbu, South Wollo, Amhara Region, a total of 144rnblood samples were collected from P. falciparum mono-infected study participants usingrnwhatman filter paper in 2005 (N=72) and 2008 (N=72). PCR based dot blot-hybridizationrntechnique was performed to determine the change in prevalence of molecular marker genes,rnPfcrt 76 Thr and Pfmdr1 86 Tyr that confer chloroquine resistance in P. falciparumrnisolates from 2005 and 2008. The study showed a slight reduction in the prevalence ofrnPfcrt 76 mutation (Lys 76 Thr) from 100% in 2005 to 98.21% in 2008 (95% CI, -0.017-rn0.053; P= 0.29), whereas, Pfmdr1 86 mutation (Asn 86 Tyr) showed a statisticallyrnsignificant reduction of 15.9%, from 81.97% in 2005 to 66.07% in 2008 (95% CI, 0.001-rn0.315; P= 0.049). The prevalence of wild allele of Pfcrt 76 Lys increased to1.79% in 2008rnfrom 0% in 2005 (95% CI,-0.053- 0.017; P= 0.29). Similarly, the wild allele of Pfmdr1 86rnAsn was also increased from 14.75% in 2005 to 23.21% in 2008 (95% CI, -0.227- 0.057;rnP= 0.24). Overall, the findings of the present study showed a decline in prevalence ofrnchloroquine resistance conferring genes in spite of use of chloroquine for treatment of P.rnvivax in the study area. This is a good indication of the re-emergence of chloroquinernsensitive P. falciparum in the study area. Therefore, it would be advisable to withdraw thernuse of chloroquine for vivax malaria treatment for a much faster re-emergence ofrnchloroquine sensitive P. falciparum parasites, in order to maximize the likelihood ofrnchloroquine reintroduction.rnKey words: Plasmodium falciparum; Chloroquine resistance; Pfcrt; Pfmdr1; PCR; Dotrnblot-hybridization.