Background: Amoebiasis, caused by Entamoeba histolytica, is a major health concern in tropical regions like India. Stool microscopy, the primary diagnostic method, has limited sensitivity due to morphological similarities with Entamoeba dispar and Entamoeba moshkovskii, an emerging pathogen. This study evaluates the effectiveness of quantitative polymerase chain reaction with high-resolution melting (qPCR-HRM) in distinguishing these morphologically similar Entamoeba species. Methods: The qPCR-HRM method was standardized using control strains of E. histolytica, E. dispar and E. moshkovskii. The assay was further evaluated on 150 stool samples, with species confirmation achieved through conventional PCR and Sanger sequencing. Results: The melting peaks of E. histolytica and E. moshkovskii were at 80±2°C and 82±2°C, respectively, and for E. dispar at 69±2°C. The qPCR-HRM was able to detect as low as 10 fg of parasitic DNA. Of 150 stool samples, a total of 10 (6.6%) were found to be positive for E. histolytica, 13 (8.6%) for E. dispar and 7 (4.6%) for E. moshkovskii. Conclusions: This study is the first to standardize qPCR-HRM for the detection and differentiation of Entamoeba species from India. The qPCR-HRM assay offers a sensitive, specific and cost-effective diagnostic tool, contributing to improved patient management. © 2025 Elsevier B.V., All rights reserved.