Hepatitis B virus (HBV) causes hepatitis, cirrhosis, liver failure, and liver cancer, but the current therapies that employ either nucelos(t)ide analogs or (pegylated)interferon α do not clear the infection in the large majority of patients. Inhibitors of the HBV ribonuclease H (RNaseH) that are being developed with the goal of producing anti-HBV drugs are promising candidates for use in combination with the nucleos(t)ide analogs to improve therapeutic efficacy. HBV is genetically very diverse, with at least 8 genotypes that differ by ≥8% at the sequence level. This diversity is reflected in the viral RNaseH enzyme, raising the possibility that divergent HBV genotypes or isolates may have varying sensitivity to RNaseH inhibitors. To evaluate this possibility, we expressed and purified 18 patient-derived RNaseHs from genotypes B, C, and D. Basal RNaseH activity and sensitivity to three novel RNaseH inhibitors from three different chemotypes were assessed. We also evaluated four consensus HBV RNaseHs to determine if such sequences would be suitable for use in antiviral drug screening. The patient-derived enzymes varied by over 10-fold in their basal RNaseH activities, but they were equivalently sensitive to each of the three inhibitors. Similarly, all four consensus HBV RNaseH enzymes were active and were equally sensitive to an RNaseH inhibitor. These data indicate that a wide range of RNaseH sequences would be suitable for use in antiviral drug screening, and that genotype- or isolate-specific genetic variations are unlikely to present a barrier during antiviral drug development against the HBV RNaseH.