AbstractB‐lymphoblastic leukemia/lymphoma (B‐ALL) is the most common pediatric malignancy and the most commonly diagnosed adult lymphoblastic leukemia. Recent advances have broadened the spectrum of B‐ALL, with DUX4 gene fusions implicated in a subclass occurring in adolescents and young adults and harboring a favorable prognosis. DUX4 fusions have been challenging to identify. We aimed to determine whether expression of the DUX4 oncoprotein, as detected by targeted immunohistochemistry, might serve as a surrogate for molecular detection of DUX4 fusions in B‐ALL. A cohort of investigational B‐ALLs was generated with enrichment for DUX4 fusions by the inclusion of cases with characteristic demographic features and immunophenotypic properties. B‐ALLs with mutually exclusive cytogenetics were collected. Immunohistochemical staining by a monoclonal antibody raised against the N‐terminus of the DUX4 protein was performed. N‐DUX4 immunohistochemistry demonstrated strong, crisp nuclear staining in blasts of seven investigational cases, six of which had nucleic acid material available for molecular evaluation. Five of these cases demonstrated RNA‐seq DUX4‐fusion positivity. One N‐DUX4 immunohistochemistry positive case lacked a definitive DUX4‐fusion by RNA‐seq, though demonstrated a gene expression profile characteristic of DUX4‐rearranged B‐ALLs, a CD2+ immunophenotype, and a lack of staining by C‐terminus DUX4 antibody immunohistochemistry. At least 83.3% [5/6] positive predictive value. N‐DUX4 immunohistochemistry was negative in blasts of three RNA‐seq DUX4‐fusion‐negative cases (3/3; 100% negative predictive value). B‐ALLs with mutually exclusive cytogenetic profiles were all N‐DUX4 negative (0/10, specificity 100%). N‐DUX4 immunohistochemistry is reliable for the distinction of DUX4‐rearranged B‐ALLs from other B‐ALLs. We recommend its use for subclassification of B‐ALLs in adolescents and young adults and in B‐ALLs that remain “not otherwise specified.”