BACKGROUNDPrimary effusion lymphoma (PEL) is a rare subtype of large B‐cell lymphoma that arises in body cavities without detectable tumor masses. PEL is universally associated with Kaposi sarcoma herpesvirus (KSHV)/human herpesvirus‐8 (HHV8). Despite overlapping features, KSHV/HHV8‐negative effusion‐based lymphoma is a distinct entity from PEL. To date, 52 cases have been reported. The authors report 3 additional cases received in their laboratory from 2007 to 2012.METHODSClinical data, cytomorphologic features, and immunophenotypic features of the 3 cases were described and compared with those reported in the literature.RESULTSThe cells in HHV8‐negative effusion lymphoma commonly revealed large cell, immunoblastic morphology and B‐cell immunophenotype. The 3 cases demonstrated cytomorphologic and immunophenotypic variability. Cytomorphologically, 1 case contained large, highly atypical cells with a moderate amount of cytoplasm, round nucleus, coarsely granular chromatin, and a single macronucleolus. The other 2 cases had medium to large atypical cells with high nuclear‐to‐cytoplasmic ratios, slightly irregular to cleaved nuclei, and multiple conspicuous nucleoli. One case had a null phenotype with aberrant cytokeratin expression. B‐cell phenotype was established by clonal immunoglobulin heavy‐chain rearrangement using polymerase chain reaction, whereas the other 2 cases demonstrated a B‐cell phenotype by flow cytometry and immunohistochemical staining. All 3 cases were negative for both HHV8 and Epstein‐Barr virus.CONCLUSIONSHHV8‐negative effusion lymphoma exhibits clinical, cytomorphologic, and immunophenotypic variability. Cases with a null‐phenotype can be particularly challenging. When effusion lymphoma is suspected, ancillary tests are helpful. Moreover, HHV8 detection is critical in differentiating PEL and HHV8‐negative effusion lymphoma, because they have overlapping features yet different prognoses. Cancer (Cancer Cytopathol) 2013;121:661–9. © 2013 American Cancer Society.