Summary In this single centre study of childhood acute lymphoblastic leukaemia (ALL) patients treated on the Medical Research Council UKALL 97/99 protocols, it was determined that minimal residual disease (MRD) detected by real time quantitative polymerase chain reaction (RQ‐PCR) and 3‐colour flow cytometry (FC) displayed high levels of qualitative concordance when evaluated at multiple time‐points during treatment (93·38%), and a combined use of both approaches allowed a multi time‐point evaluation of MRD kinetics for 90% (53/59) of the initial cohort. At diagnosis, MRD markers with sensitivity of at least 0·01% were identified by RQ‐PCR detection of fusion gene transcripts, IGH/TRG rearrangements, and FC. Using a combined RQ‐PCR and FC approach, the evaluation of 367 follow‐up BM samples revealed that the detection of MRD >1% at Day 15 (P = 0·04), >0·01% at the end of induction (P = 0·02), >0·01% at the end of consolidation (P = 0·01), >0·01% prior to the first delayed intensification (P = 0·01), and >0·1% prior to the second delayed intensification and continued maintenance (P = 0·001) were all associated with relapse and, based on early time‐points (end of induction and consolidation) a significant log‐rank trend (P = 0·0091) was noted between survival curves for patients stratified into high, intermediate and low‐risk MRD groups.