<p>To explore the relevance of non-malignant B cells (NMBCs) in chronic lymphocytic leukaemia (CLL), 201 blood samples from 79 previously untreated CLL patients receiving chemoimmunotherapy (CIT) in a phase III clinical trial were subjected to mass cytometry analysis using a bespoke panel of 24 antibodies. One memory (CD27⁺CD38⁻) and two naïve (CD27⁻CD38⁺) CD19⁺ clusters with a non-malignant phenotype (NMP; CD5⁻CD43⁻ROR1⁻CD20^hiCD79b^hiCD81⁺) were identified by FlowSOM and UMAP algorithms. NMP clusters were most prevalent in healthy controls, almost undetectable in untreated and relapsed CLL, and variable following CIT, where they correlated with haematopoietic recovery (naïve NMP clusters) and polyclonal antibody production (memory NMP cluster). Larger NMP cluster size at the early post-treatment timepoint was associated with a significantly longer progression free survival (PFS; HR 0.40 [95% CI: 0.22–0.75]; <i>P</i> = 0.003). Similar findings were obtained when NMBCs were prospectively defined by Boolean gating using the non-redundant features of the NMP clusters. NMBCs measured in this way negatively correlated with MRD and provided complementary prognostic information in patients with persistent MRD. Our findings raise the possibility that re-emergence of circulating NMBCs after treatment reflects clearance of CLL from secondary lymphoid organs, thereby complementing MRD as a biomarker of bone marrow clearance.</p>