Abstract Essential NK cell-mediated murine CMV (MCMV) resistance is under histocompatibility-2k (H-2k) control in MA/My mice. We generated a panel of intra-H2k recombinant strains from congenic C57L.M-H2k/b (MCMV resistant) mice for precise genetic mapping of the critical interval. Recombination breakpoint sites were precisely mapped and MCMV resistance/susceptibility traits were determined for each of the new lines to identify the MHC locus. Strains C57L.M-H2k(R7) (MCMV resistant) and C57L.M-H2k(R2) (MCMV susceptible) are especially informative; we found that allelic variation in a 0.3-megabase interval in the class I D locus confers substantial difference in MCMV control phenotypes. When NK cell subsets responding to MCMV were examined, we found that Ly49G2+ NK cells rapidly expand and selectively acquire an enhanced capacity for cytolytic functions only in C57L.M-H2k(R7). We further show that depletion of Ly49G2+ NK cells before infection abrogated MCMV resistance in C57L.M-H2k(R7). We conclude that the MHC class I D locus prompts expansion and activation of Ly49G2+ NK cells that are needed in H-2k MCMV resistance.