Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although the cure rate of ALL has greatly improved, a considerable number of patients suffer from relapse of leukemia. Therefore, ALL remains the leading cause of death from cancer during childhood. To improve the cure rate of these patients, precisely detecting patients with high risk of relapse and incorporating new targeted therapies are urgently needed. This study investigated inexpensive, rapid, next-generation sequencing of more than 150 cancer-related genes for matched diagnostic, remission, and relapse samples of 17 patients (3 months to 15 years old) with relapsed ALL. In this analysis, we identified 16 single-nucleotide variants (SNVs) and insertion/deletion variants and 19 copy number variants (CNVs) at diagnosis and 28 SNVs and insertion/deletion variants and 22 CNVs at relapse. With these genetic alterations, we could detect several B cell precursor ALL patients with high-risk gene alterations who were not stratified into the highest-risk group (5/8, 62.5%). We also detected potentially actionable genetic variants in about half of the patients (8/17, 47.1%). Among them, we found that one patient harbored germline TP53 mutation as a secondary finding. This inexpensive, rapid method can be immediately applied as clinical sequencing and could lead to better management of these patients and potential improvement in the survival rate in childhood ALL.