Approximately half of patients with chronic myeloid leukemia (CML) in sustained deep molecular response who discontinue tyrosine kinase inhibitors (TKIs) remain in treatment-free remission (TFR). Some of these patients have measurable residual disease (MRD) by BCR-ABL1 mRNA testing, and most have detectable BCR-ABL1 DNA by highly sensitive methods. We used fluorescence-activated cell sorting and BCR-ABL1 DNA PCR to investigate the lineage of residual CML cells in TFR. Twenty patients in TFR for >1 year provided blood for sorting into granulocytes, monocytes, B cells, T cells, and NK cells. MRD was identified predominantly in the lymphoid compartment and never in granulocytes. B cells were more often BCR-ABL1 positive than T cells (18 vs 11/20 patients) and at higher levels (median 10-4.9 vs 10-5.7; P = 0.014). In 13 CML patients studied at diagnosis lymphocytes expressing BCR-ABL1 mRNA comprised a small proportion of total leukocytes. These data improve our understanding of TFR biology, since it is now clear that MRD in the blood of TFR patients need not imply the persistence of multipotent CML cells. Lineage-specific assessment of MRD could be explored as a means to improve the prediction of TFR.