Objectives: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. Methods: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. Results: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P =.011), ELN adverse risk (P =.002), NPM1 wild type (WT, P =.049), FLT3 WT (P =.035), and relatively low percentages of leukemia-associated immunophenotypes (P =.006). Additional immunophenotyping analysis revealed the CLL-1− subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1− subfractions of bimodal samples (N = 3). Conclusions: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1− cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation.