Natural Killer (NK) cell recognition of the non-classical Human Leukocyte Antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus (CMV) can manipulate this central innate interaction through the provision of a mimic of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here we analysed UL40 sequences isolated from 32 hematopoietic stem cell transplantation (HSCT) recipients experiencing CMV reactivation. The UL40 protein showed a polymorphic hotspot within the region that encodes the HLA leader sequence mimic. While all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.