De novo diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide, accounting for approximately 30–40% of adult non-Hodgkin lymphomas. It is defined as a diffuse proliferation of large neoplastic B lymphoid cells with a nuclear size equal to or exceeding that of a normal macrophage or more than twice the size of a normal lymphocyte. As such, de novo DLBCL is heterogeneous in morphology, immunophenotypic features, and biologic behavior, including response to chemotherapy and ultimate outcome. In this chapter, we discuss the applications of gene expression profiling (GEP) to this large, heterogeneous group of lymphoma, in terms of diagnosis, differentiation from other B-cell lymphomas, and biologic behavior.