Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and the leading cause of childhood death in contrast to the 90% cure rate. ALL includes different subtypes described by interrupt collections of somatic chromosomal alterations and sequence mutations that disrupt normal body functions such as lymphoid maturation, cell-cycle regulation, and tumor suppression. Having a significant role in several cancers, the high mobility group box-1 (HMGB1) gene is considered an important gene in the development of tumors. Methods: Herein, the genetic role of HMGB1 was studied in the 49 Iranian patients with newly diagnosed ALL using Sanger sequencing of HMGB1 coding regions (exons 2 to 5). Results: The results showed that none of the subjects in the study had any promising variants in the coding sequences of the HMGB1. Conclusion: These findings suggest that HMGB1 is not directly associated with ALL incidence and behavior. Further investigations using a large group of patients with different races and ethnicities are required to analyze the possible role of HMGB1 gene polymorphisms in ALL patients.