► The Tm of a TaqMan probe employed in a widely used RT-qPCR for HEV was calculated. ► Calculations showed that the melting temperature (Tm) of the probe was suboptimal. ► A Minor Groove Binder version of probe was synthesised in order to increase its Tm. ► MGB probe detected HEV in 6 samples that were negative with the unmodified probe. ► False negative results were due to a C→T point mutation in the probe binding region. Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the developing world. It is responsible for both sporadic infections and large scale epidemics and may be associated with significant mortality during pregnancy. Over the past two decades many serological and nucleic acid based diagnostic tests for HEV have been developed, including several reverse transcription real-time polymerase chain reaction assays (RT-qPCR). One of the most widely used of these RT-qPCRs is that developed by Jothikumar and colleagues (Journal of Virological Methods 2006, 131, 65–71). Whilst reviewing this assay we calculated the predicted melting temperature of its TaqMan probe and consequently synthesised a minor groove binder (MGB) version in order to increase its hybridisation stability. In this report the performance of the original unmodified probe is compared with that of the MGB-modified version. We demonstrate that the MGB-modified probe detected HEV RNA in plasma samples from six patients with serologically confirmed hepatitis E in whom the unmodified probe had failed to detect HEV RNA. Sequence analysis of the ORF3 segment targeted by the RT-qPCR was possible in 4 of the 6 patients and revealed an identical C→T single nucleotide mutation in the probe binding region in each case.