Hepatitis E virus (HEV) is an emerging public health concern in Saudi Arabia, yet its molecular epidemiology remains poorly characterized. The current study aims to investigate HEV genotypes circulating in the West Riyadh region through phylogenetic analysis of clinical samples. Molecular epidemiological study was conducted over the two-years period 2022-2023 at the Blood Donation Center of Dawadmi General Hospital (BDCDGH). Out of 245 samples that tested positive for either IgM or IgG antibodies against HEV were selected for further molecular analysis. Only 0.8% (2/245) of ELISA-positive samples were confirmed HEV RNA-positive by PCR. This 0.8% PCR positivity reflects the natural resolution of most HEV infections in immunocompetent hosts, with only acute cases detectable by PCR, while ELISA captures both past and present exposures. Sequencing identified 2 distinct genotypes, HEV-1a (Dawadmi_Isolate_02) showing 99.7% nucleotide identity with Burmese reference strains, suggesting imported transmission, and HEV-3b (from an Indian worker) closely related (98.06%) to Japanese/Nepalese strains, indicating potential zoonotic or travel-associated exposure. The HEV-1a isolate contained characteristic neutralization epitopes (⁴⁵⁷SGPSLTPF⁴⁶⁴) and proline-rich domains, while the HEV-3b strain harbored subtype-specific mutations (S563L, V617A). Demographic analysis revealed a male-dominated (96%), middle-aged (88% aged 41-60 years) cohort, with unexpected occupational distribution (70% clerks). These findings demonstrate the co-circulation of non-endemic HEV strains in Saudi Arabia, highlighting gaps in current surveillance systems. The study underscores the need for enhanced screening of high-risk groups, zoonotic monitoring, and genomic-surveillance to guide prevention strategies in alignment with Saudi Vision 2030 health objectives.

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