Genomic studies, such as gene expression profiling and next-generation sequencing studies, have provided new insights into the phenotypic characteristics and pathogenesis of mature aggressive B-cell lymphomas. In particular, mutations in the transcription factors ID3 and TCF3, leading to overexpression of B-cell receptor components such as VPREB3, have been shown to be specific for Burkitt lymphoma (BL) and play an important tumourigenic role by mediating the activation of the pro-survival phosphatidylinositol-3-OH kinase pathway. We performed immunohistochemical analysis by applying commercially available anti-VPREB3 antibody to a large cohort of 185 genetically and immunophenotypically characterized mature aggressive B-cell lymphomas and analyzed these results together with recent data on ID3 expression. The combined expression of both VPREB3 and ID3 was associated with a diagnosis of BL with high sensitivity (0.77), high specificity (0.75) and high negative predictive values (0.96), however, with lower positive predictive value (0.30). Double negative cases were absent in the group of BLs but could be found in approximately one third of the remaining cases of mature aggressive B-cell lymphomas. Further, we could not identify a correlation with MYC, BCL2 or BCL6 aberrations with neither VPREB3 nor ID3 expression in each of the diagnostic groups analyzed. Our results, which are in line with recently discovered mutations in next-generation sequencing studies, suggest that the combined immunohistochemical detection of VPREB3 and ID3 is applicable to the routine diagnostic in case of mature aggressive B-cell lymphomas. In particular, it represents a useful and routinely applicable diagnostic tool to exclude BL diagnosis in case of single positive or double negative cases.