Immunocompromised individuals, including those infected with human immunodeficiency virus (HIV), are at increased risk of Epstein–Barr virus (EBV)‐associated aggressive B cell malignancies such as Burkitt’s lymphoma (BL) or diffuse large B cell lymphoma (DLBCL). Differential diagnosis of these lymphomas requires histopathological, immunohistochemical and cytogenetic assessments. Rapid, less invasive approaches to the diagnosis of EBV‐associated B cell lymphomas are needed. Here, high‐throughput cytokine profiling of BL cell lines and EBV‐transformed B lymphoblastoid cell lines (B‐LCL), representing DLBCL, was carried out. By monitoring the production of 42 different cytokines, unique cytokine signatures were identified for BL and B‐LCL/DLBCL. The BL cells produced interleukin (IL)‐10, 10 kDa interferon gamma‐induced protein (IP‐10)/CXCL10, macrophage‐derived chemokine (MDC)/CCL22, macrophage inflammatory protein (MIP)‐1α/CCL3 and MIP‐1β/CCL4. In addition to these five cytokines, the cytokine signature of B‐LCL/DLBCL cells included IL‐8/CXCL8, IL‐13, platelet‐derived growth factor (PDGF)‐AA, and regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5. Epstein–Barr virus latency was responsible for the increased production of IL‐10, MDC/CCL22 and MIP‐1α/CCL3 in BL cells, suggesting that EBV‐mediated BL‐genesis involves these three cytokines. These results suggest that high‐throughput cytokine profiling might be a valuable tool for the differential diagnosis and might deepen our understanding of the pathogenesis of EBV‐associated B cell malignancies. (Cancer Sci 2011; 102: 1236–1241)