CD26 + leukemic stem cells (LSC) are a specific marker for chronic myeloid leukemia (CML), absent in healthy individuals and other myeloid neoplasms. These cells can contribute to disease resistance, as they are believed to sustain the leukemic clone despite effective tyrosine kinase inhibitor (TKI) therapy. This study analyzed CD26 + LSC and BCR::ABL1 transcript levels simultaneously using multiparametric flow cytometry and RT-qPCR in 210 chronic-phase patients undergoing TKI therapy and 31 patients in treatment-free remission (TFR). A significant decrease in LSC levels was observed as patients achieved deep molecular response (DMR, BCR::ABL1IS ≤ 0.01%) (χ2, p < 0.001). However, 19% (14/73) of DMR patients displayed persistent CD26 + LSC, suggesting a quiescent state without detectable BCR::ABL1 transcripts. A weak correlation (r = 0.187, p = 0.046) between LSC/µL absolute number and BCR::ABL1 transcript levels indicates a limited predictive value between these two variables. In TFR patients, LSC recurrence during follow-up did not correlate with molecular relapse, questioning their clinical relevance in this setting. In conclusion, while CD26 + LSC are frequently observed in patients with poor molecular response, their levels significantly decrease as patients achieve DMR. However, their persistence or recurrence in TFR lacks prognostic value for molecular relapse, indicating that CD26 + LSC are not reliable predictors of outcomes in CML.