Aims: to determine the relative utility of in situ testing for hepatitis E virus (HEV) RNA and paraffin section PCR to diagnose HEV infection in paraffin-embedded clinical liver biopsies, and to correlate with clinico-pathological characteristics. Methods and results: We evaluated in situ and quantitative PCR (qPCR)-based approaches to identifying HEV in clinical liver biopsies from infected patients from multiple centers, correlating with clinical setting (immunocompetent, allograft or immunosuppressed native liver) and histologic findings. 36 biopsies from 29 patients had histologic data, of which 27 and 23 biopsies had satisfactory material for in situ RNA testing and tissue qPCR respectively. Both approaches specifically identified HEV infection, but tissue qPCR was significantly more sensitive than in situ testing (P=0.035). In immunocompetent but not immunosuppressed patients the tissue qPCR yield correlated with the severity of lobular hepatitis (rho=0.94, P<0.001). qPCR viral yield was comparably high in allografts and immunosuppressed native livers and significantly greater than with native liver infection. Immunosuppressed patients showed reduced severity of hepatitis and cholestatic changes, compared with immunocompetent patients. Indeed, HEV-infected liver allografts could show minimal hepatitis for many months. In individual cases each technique was useful when serum was not available to retrospectively identify chronic infection (in biopsies taken 4-31 months before diagnosis), to identify persistent/residual infection when contemporary serum PCR was negative and to identify cleared infection. Conclusions: qPCR is better than in situ RNA testing to identify HEV infection in paraffin-embedded liver biopsies and has diagnostic utility in selected settings.