Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and the leading cause of childhood death in contrast to the 90% cure rates. 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 considered an important gene in the development of tumors. 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). The results showed that none of the subjects in the study had any promising variants in the coding sequences of the HMGB1. 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.