Little is known about B-lymphoblastic leukemia (B-ALL) that lacks expression of terminal deoxynucleotidyl transferase (TdT). To address this, we performed the largest study to date of TdT-negative B-ALL using data from St. Jude Total XV and XVI clinical trials. Compared to TdT-positive B-ALL (n = 896), TdT-negative B-ALL (n = 21) was associated with younger age (median, 1.4 versus 6.8 years, P < 0.001), higher white blood cell count (median, 52.8 versus 9.9 × 109/L, P < 0.001), absence of hyperdiploidy (0 versus 27.8%, P = 0.002), KMT2A rearrangement (100 versus 1.9%, P < 0.001), and inferior 5-year event-free survival (EFS) (76.2 versus 90.3%, P = 0.047). In the context of KMT2A-rearranged B-ALL (n = 38), TdT-negativity was significantly associated with the MLLT1 rearrangement partner (P = 0.026) but was not independently predictive of survival, suggesting that the high-risk features of TdT-negative B-ALL are secondary to underlying KMT2A rearrangements. Finally, we compared the sensitivity of TdT-negativity to neuron-glial antigen 2 (NG.2) expression for the detection of KMT2A rearrangements and found that 63% of KMT2A-rearranged B-ALL cases not identified by NG.2 were TdT-negative. The results of this study expand the spectrum of immunophenotypic features that are specific for high-risk KMT2A rearrangements in pediatric B-ALL and can be readily implemented using existing standard acute leukemia flow cytometry panels.