Background:Telomere length maintenance is tightly regulated with cell proliferation and senescence. To overcome physiologic telomere attrition with cell division, long‐term repopulating cells like stem and progenitor cells upregulate telomerase. Pathophysiologically, over 90% of cancer cells reactivate telomerase enabling replicative immortality. Acute lymphoblastic leukemia (ALL) is the most common type of acute leukemias in pediatric patients (pts) and a neoplastic disease with a rapid clonal expansion.Aims:We aimed to assess the mitotic history of the malignant ALL clone by telomere length (TL) and the potential of telomerase activity (TA) as a prognostic marker.Methods:Peripheral blood samples collected before treatment initiation from 9 pediatric pts (4 females/ 5 males) with newly diagnosed pre‐B cell ALL according to WHO 2016 criteria were analyzed for TL and TA. All pts were treated according to the international collaborative protocol (AIEOP‐BFM ALL 2009 Registry) for children and adolescents with ALL. Based on (cyto)genetics and treatment response, 6 pts are in the intermediate and 3 pts are in the high‐risk prognostic group. TL was measured in subsets of leukocytes by automated multicolor flow‐FISH and compared to TL percentiles of the reference range obtained from over 400 healthy individuals. TA in leukocytes was analyzed by Telomere Repeat Amplification Protocol.Results:The median age of the patient (pt) cohort was 10.8 years (range: 2.8–17.8) at diagnosis and study entry. The median leukocyte count was 9.8 G/L (1.4–38.6) with a median lymphocyte count of 2.0 G/L (0.6–8.1) and a median lymphoblast count of 6.37 G/L (0.2–31.27). The median TL value in blasts was with 5.2 kb (1.9–8.3) significantly (p = 0.016) lower than 7.5 kb (6.9–9.3) for the B‐cell subset. In 6/9 pt samples the TL values for blasts were below the 1st percentile of the TL reference range for lymphocytes whereas 3 pts had values between the 1st and 50th percentile. The median age‐adjusted difference in TL (dTL = difference of TL to the 50th percentile for respective age) was −4.5 kb (−7.1–1.1) in blasts which was significantly higher than median dTL values in lymphocytes (−2.1 kb; range −6.2–0) and B‐cells (−1.9 kb, range −2.6–0.4). Median TA in the mononuclear cell fraction containing blasts was 2.4 telomere‐elongated products (TP)/cell (0.3–11.1), whereas no TA was detected in leukocytes from healthy donors. Notably, pediatric ALL pts with a high risk score (n = 3) had a higher median TA of 5.5 TP/cell (2.2–11.1) than those with an intermediate risk score (n = 6) with 1.8 TP/cell (0.3–3.3).Summary/Conclusion:The significantly lower TL values in ALL blast cells from newly diagnosed pediatric pts reflect the mitotic history of the malignant clone with a high number of cell divisions. Despite measurable TA in ALL blasts, attrition of TL seemed not compensated and therefore, the extent of clonal proliferation might even be underestimated. Importantly, the higher median TA in blast cells of pts in the high‐risk prognostic group indicates that TA could be an attractive prognostic marker. Further data on TL and TA on serial pt samples will identify if TA could also serve as predictive marker and therapeutic target.