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Objective: This study aimed to investigate the prevalence and subtype distribution of the genus Blastocystis using high-resolution melting curve analysis (HRM), a novel molecular technique. Blastocystis sp., a unicellular, anaerobic, and zoonotic parasite, is commonly found in the digestive tracts of humans and animals. Transmission occurs via the fecal-oral route, with prevalence rates ranging from 20% in developed countries to over 60% in developing regions. Although the pathogenicity of Blastocystis remains debated, it has been detected in both symptomatic and asymptomatic individuals. Traditionally, direct microscopy with wet mounts is used for the rapid identification of Blastocystis in stool samples. However, molecular diagnostics have become essential for the accurate detection and differentiation of Blastocystis subtypes MATERIALS AND METHODS: We collected 730 stool samples from humans and domestic animals. Initial screening for Blastocystis sp. was performed using direct microscopy. Negative samples were cultured in a two-phase culture medium and re-examined after 2-3 days. HRM analysis was then employed to identify and differentiate Blastocystis subtypes using specific primers via real-time PCR., Results: Six subtypes were identified, with ST7 (30%) being the most prevalent, followed by ST3 (28%), ST2 (16%), ST1 (14%), ST5 (6%), and ST14 (6%). In humans, ST3 was the most common subtype, also found in poultry and sheep, whereas ST7 was mainly detected in domestic animals. Notably, ST1-ST3 were identified in domesticated animals, indicating a pattern of cross-species transmission., Conclusion: ST3 (28%) and ST7 (30%) were the most prevalent subtypes across all samples, with distinct distributions in human and animal hosts. The HRM technique demonstrated efficiency and cost-effectiveness, providing a rapid and accurate method for Blastocystis subtype identification in developing countries, which can expedite diagnostic responses and reduce the need for sequencing. This supports the potential for HRM to improve epidemiological surveillance and understanding of cross-species transmission., Competing Interests: Declarations. Ethical approval: This study was approved by the Ethical Committee of Mashhad University of Medical Sciences (IR.MUMS.MEDICAL.REC.1400.328), in accordance with the Helsinki Declaration and ethical guidelines. The project was part of the thesis titled “Thesis number: 992196.” During the study, we only accessed anonymized samples, with all personal identifying information, including names, addresses, and phone numbers, removed before the samples were provided to the research team. Consequently, we did not have access to any personally identifiable information either during or after data collection. The research adhered to strict ethical standards to ensure the privacy and confidentiality of all participants. Consent to participate: In this study, we utilized daily and weekly archived stool samples from patients who attended our medical centers. These samples were provided with a coding system, and we did not have access to any identifying information, such as the patients’ names, addresses, or phone numbers. As a result, we did not obtain informed consent directly from the patients, as their personal identities were not involved in the research process. The ethical committee reviewed and approved the use of these anonymized samples, waiving the requirement for informed consent due to the absence of identifiable information. Consent for publication: All participants and institutions involved in this study provided consent for the publication of the anonymized results. Clinical trail: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)