Objective: To explore the characteristics and clinical significance of clonal heterogeneity in patients with acute lymphoblastic leukemia(ALL). Methods: From January 2016 to June 2019, 170 newly diagnosed ALL patients were enrolled in the Department of Hematology, Henan Cancer Hospital, including 93 males and 77 females, with a median age of 17 (2-80) years. Fifty-two ALL-related genes were detected by high-throughput sequencing technique. The clonal heterogeneity of mutations was analyzed according to the variant allele frequency (VAF) and the results of flow cytometry. The prognostic value of mutations was also evaluated. Results: Gene mutations were detected in 121 (71.2%, 121/170) patients, of which 2 or more clones were detected in 18 (52.9%, 18/34) T-cell acute lymphoblastic leukemia patients, while only 23 (16.9%, 23/136) B-cell acute lymphoblastic leukemia patients were positive of multiple mutations (P<0.01).Gene mutation-related clonal heterogeneity analysis showed that 2 or more clones were frequent in patients with NOTCH1 mutations (13/19 patients) (P<0.01). Event free survival (EFS) in patients with 3 or more clones was significantly lower than other patients (χ(2)=10.330, P=0.016). Child ALL patients had similar result, that multiple clones predicted lower overall survival (OS) and EFS (OS: χ(2)=7.974, P=0.047; EFS: χ(2)=10.860, P=0.013). Conclusion: Clonal heterogeneity in ALL patients is closely related to the different origin of lymphocyte lineages and the age of onset, which may reveal the nature of the disease and predict the clinical outcome.