Infectious complications represent the second most common cause of mortality and a major cause of morbidity in β-thalassemia major (BTM), with a prevalence of 12–13%. The data on unconventional T-lymphocyte subsets in BTM children are limited. The aim of the present study was to investigate and evaluate phenotypic alterations in CD4+CD8+ double positive (DP), CD4− CD8− double negative (DN), and natural killer T-lymphocytes (NKT) in BTM children in comparison to healthy controls. Our case control study included 80 children with BTM and 40 healthy children as controls. Assessment of unconventional T-lymphocyte populations was done using sensitive fourcolor flow cytometry (FACSCalibur). Our analysis of the data showed a significantly higher frequency CD4+ CD8+ (double-positive) T cells, CD4− CD8− (double negative) T cells, and natural killer T cells in the peripheral blood of both BTM groups (splenectomized and non-splenectomized) as compared to healthy controls, suggesting that these cells may play a role in the clinical course of BTM. The relationship of the unconventional T-lymphocytes to immune disorders in BTM children remains to be determined. Further longitudinal study with a larger sample size is warranted to elucidate the role these cells in BTM. UMIN‑CTR study design: trial number UMIN000018950.