Background: Allogenic hematopoietic stem cell transplantation (HSCT) is a strategy used for treatment of different malignant diseases. However, success of allo-HSCT can be hampered by graft-versus-host-disease (GVHD). Natural killer (NK) cells may play an important role in activating antigen presenting cells and subsequent activation of T cells. The main purpose of this study was the evaluation of IL-21, as a blood biomarker, for early detection of acute GVHD (aGVHD) in children after HSCT and also the study of human leukocytes antigen (HLA)-C1 polymorphism, as a targeting ligand for NK cells in these patients. Methods: Fifty one children receiving HSCT were studied. Blood samples were collected at -8, 7, and 14 days of transplantation. The -8-day samples were analyzed for HLA-C1 polymorphism by PCR-sequence-specific primer technique and pre-transplantation IL-21 assay. To study the serum levels of IL-21, two blood samples were collected on days +7 and +14 and analyzed by ELISA technique. Results: The results indicated that the incidence of aGVHD in pediatric is associated with a polymorphism of HLA-C1, as alleles HLA-C01:12 (P<0.001), HLA-C01:22 (P<0.004), and HLA-C01:67 (P<0.009). On the other hand, the serum levels of IL-21 in children with aGVHD were decreased after transplantation compared to before transplantation. The serum levels of the IL-21 at 14 days after transplantation had a significant correlation with the occurrence of aGVHD (P=0.05). Conclusion: Based on the findings of this study, there is a significant correlation between HLA-C1 polymorphisms and the serum levels of IL-21 with the incidence of aGVHD.