In conclusion, our newly-developed PCR-RFLP method demonstrated that differential restriction patterns could be experimentally obtained from the three morphologically indistinguishable species of Entamoeba. Research is underway in Honduras and Nicaragua to validate this protocol using stool samples from symptomatic patients. Once successfully implemented, this method will help advance amoebiasis clinical diagnosis and management as well as research in the region. Additionally, it will enable the investigation of E. moshkovskii, a species that, to our knowledge, has yet to be described in Central America. Most importantly, our new method can be a reliable option to help determine the true prevalence of Entamoeba histolytica infections in Honduras and Nicaragua.