Background: The finding of many Thai Hb E-β0-thalassemia patients with non-transfusion dependent thalassemia (NTDT) phenotype without co-inheritance of α-thalassemia has prompted us to investigate the existence of other genetic modifying factors. Methods: Study was done on 146 adult Thai patients with NTDT Hb E-β0-thalassemia and a homozygous β-thalassemia patient without co-inheritance of α-thalassemia. Multiple single-nucleotide polymorphisms (SNPs) associated with γ-globin gene expression including the Gγ-XmnI of HBG2 gene, rs2297339, rs4895441, and rs9399137 of the HBS1L-MYB gene, rs4671393 in the BCL11A gene, and G176AfsX179, T334R, R238H and -154 (C-T) in the KLF1 gene were investigated using PCR-and related techniques. Results: Heterozygous and homozygous for Gg-XmnI of HBG2 gene were detected at 68.0% and 6.1%, respectively. Further DNA analysis identified the rs2297339 (C-T), rs4895441 (A-G), and rs9399137 (T-C) of HBS1L-MYB gene in 86.4%, 22.5% and 20.4%, respectively. The rs4671393 (G-A) of the BCL11A gene was found at 31.3%. For the KLF1 gene, the T334R and G176AfsX179 (+/-) were detected at 8.2% and 1.4%, respectively. Conclusion: It was found that these SNPs when analyzed in combination could explain the mild phenotypic expression of all cases. These results underline the importance of these informative SNPs on phenotypic expression of Hb E-β-thalassemia patients.