Background: Malaria incidence has declined in Ethiopia in the past ten years. Current malariarndiagnostic tests, including light microscopy and antigen-detecting rapid tests (RDTs) cannotrnreliably detect low-density infections. Studies have shown that nucleic acid amplification testsrnare highly sensitive and specific in detecting malaria infection. Thus, this study took place withrnthe aim of evaluating the performance of multiplex real time PCR for the diagnosis of malariarnusing patient samples collected from health facilities located at malaria elimination targeted lowrntransmission settings in Ethiopia. rnMethods: A health facility based cross sectional survey was conducted in selected malariarnsentinel sites. Malaria suspected febrile outpatients referred to laboratory for malaria testingrnbetween December 2019 and March 2020 were enrolled into this study. Socio demographicrninformation and capillary blood samples were collected from the study participants and tested atrnspot with RDTs. Additionally, five circles of dry blood sample (DBS) samples on Whatman filterrnpaper and thick and thin smear were prepared for molecular testing and microscopicrnexamination, respectively. Multiplex real time PCR assay was performed at EPHI malariarnlaboratory. The performance of multiplex real time PCR assay, microscopy and RDT for therndiagnosis of malaria was compared and evaluated against each other.rnResults: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as P.rnfalciparum infection, 16 as P. vivax and 3 as mixed infections. Of the total samples, lightrnmicroscopy detected 33 as Pf, 18 as PV and RDT detected 43 as Pf, 17 as PV, and one mixedrninfection. Using light microscopy as reference test, the sensitivity and specificity of multiplexrnreal time PCR were 100% (95% CI [93-100]) and 83.2% (95% CI [77.6-87.9]), respectively.rnUsing multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58%rn(95% CI [46.9-68.4] and 67% (95% CI [56.2-76.7]); and specificity of 100% (95% CI [98-100]rnand 98.9 (95% CI (96-99.9), respectively. Substantial level of agreement was reported betweenrnmicroscopy and multiplex real time PCR results with kappa value of 0.65. rnConclusions: Multiplex real time PCR had an advanced performance in parasite detection andrnspecies identification on febrile patientsâ€™ samples than did microscopy and RDT in low malaria rntransmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria rnelimination program, particularly for community based epidemiological samples. Althoughrnmicroscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinicalrnfacilities.