Studies in T‐cell acute lymphoblastic leukemia (T‐ALL) have shown that leukemic blast populations may display immunophenotypic heterogeneity. In the clinical setting, evaluation of measurable residual disease during treatment and follow‐up is highly dependent on knowledge of the diversity of blast subsets. Here, we set out to evaluate whether variation in expression of the blast marker, TdT, in T‐ALL blasts could correspond to differences in morphometric features. We investigated diagnostic bone marrow samples from six individual T‐ALL patients run in parallel on imaging flow cytometry (IFC) and conventional flow cytometry (CFC). Guided by the imagery available in IFC, we identified distinct TdTnegand TdTpossubpopulations with apparent differences in internal complexity. As TdTnegblasts predominantly displayed very low forward scatter (FSC) on CFC, these subsets were initially excluded from routine analysis as debris, elements of small diameter, apoptotic, and/or dead cells. However, IFC‐based morphometric analyses demonstrated that cell size and shape of TdTnegblasts were comparable to the TdTposcells and without morphometric apoptotic hallmarks, supporting that the TdTnegsubpopulation corresponded to T‐ALL blasts. Fluorescence in situ hybridization analyses substantiated the clinical relevance of TdTnegFSCvery‐lowcells by retrieving known diagnostic cytogenetic abnormalities at comparable frequencies in purified TdTnegFSCvery‐lowand TdTposFSCintsubsets. We highlight this finding as knowledge of phenotypic heterogeneity is of crucial importance in the clinical setting for delineation and quantification of blast subpopulations of potential biological relevance. We argue that the IFC imagery may allow for visual verification and improvement of applied gating strategies.