Aggressive natural killer cell leukemia (ANKL) is a systemic NK-cell neoplasm, almost always associated with EBV. Rare cases of EBV-negative ANKL have been described and some reports suggested more indolent behavior. We report the clinicopathologic, immunophenotypic and molecular characteristics of seven EBV-negative ANKL. All patients were adults, with median age of 63 years (range 22–83) and a M:F ratio of 2.5:1. Five patients were White, one Black and one Asian. All patients presented acutely, with fever (6/7), cytopenias (6/7) and splenomegaly (4/7). Four patients had lymphadenopathy, 4 had extranodal disease. Bone marrow involvement was present in 5, with hemophagocytosis in 3. Peripheral blood was involved in 4 with the neoplastic cells containing prominent azurophilic granules. By immunohistochemistry and/or flow cytometry, the tumor cells lacked surface CD3, and were positive for CD56 (7/7), CD2 (5/5), CD8 (3/7), CD30 (4/5) and Granzyme B (6/6). They were negative for CD4, CD5, Beta F1, TCRγ, LMP1 and EBER. PCR for TCRG clonality was polyclonal. Mutational analysis revealed missense mutations in the STAT3 gene in both cases studied. Median survival was 8 weeks from the onset of disease. One patient received allogeneic BMT and is alive with no disease (follow-up 15 months). EBV-negative ANKL, exists, but is rare. It tends to occur in older patients and is indistinguishable clinically and pathologically from EBV-positive ANKL, with a similar fulminant clinical course. The high prevalence of Asian patients seen with EBV-positive disease seems less evident with EBV-negative cases.