Background: The Recombinant Polymerase Amplification (RPA) represents an isothermal version of the conventional polymerase chain reaction (PCR), being ideal for the detection of Leishmania DNA, especially in field conditions, as it is easy to transport, use, fast and cheap and also because with the RPA the subjectivity of the result is eliminated associated with the Direct exam.Methods: A prospective and cross-sectional study for the evaluation of the diagnostic accuracy of RPA test was conducted in different point of care of 11 municipalities of Colombia and in the reference center - PECET - Program for the Study and Control of Tropical Diseases.Findings: Samples of 128 patients with suspected CL were included and processed for analysis by Direct examination - smear, conventional PCR and RPA. The RPA diagnostic test performed at the Evaluation Center proved to be more sensitive 90.4% (95% CI 81.9 - 95.7) than the traditional Direct Exam (42 - 67%), with 14% failures in the detection of LC (false positives), without adjusting by imperfect reference standard. Similarly, RPA was more specific 72.7% (95% CI 57.2 - 85.0), with 20% of LC cases that were not detected (false negatives), without adjusting by the imperfect reference standard.Interpretation: It is justified to perform the RPA in the field not only for having a higher sensitivity than that obtained in the reference center but also because the RPA eliminates the subjectivity of the reference standard “Direct Exam”.Funding Statement: This work received financial support of the Colombian Administrative Department of Science, Technology and Innovation - Colciencias (Code: 617499847785; CT-695-2014).Declaration of Interests: The authors have declared that no competing interests exist.Ethical approval of the study was obtained from the Ethics Committee of the Faculty of Health, CES University (Act 85 of September 14th 2015). The patients included in this study signed an informed consent in which they authorized for the use of clinical samples for diagnostic research purposes. Each participant in the study was identified by a code with the finality of maintaining the confidentiality.