<p>Beta-thalassemia (β-thalassemia) is one of the most common inherited hemoglobinopathy caused by mutations in the <i>HBB</i> gene and presents with a wide clinical spectrum ranging from asymptomatic to transfusion-dependent forms. We reported the first documented case of the rare Hb Oslo variant [HBB:c.127T &gt;A; β42(CD1)Phe→Ile] in an Indian family and only the second reported case of Hb Oslo globally. Homology-based 3D structural modeling validated by Ramachandran plot, VERIFY3D, and PROCHECK indicated that Hb Oslo variant disrupts key hydrophobic interactions within the β-globin heme pocket, likely compromising heme stabilization and oxygen-binding efficiency. In addition to Hb Oslo variant, we identified the IVS1–5G &gt;C [HBB:c.92+5G &gt;C] splicing mutation in two siblings from a single Indian family, resulting in a novel compound heterozygous combination of two <i>HBB</i> gene mutations: IVS1–5G &gt;C and Hb Oslo [HBB:c.127T &gt;A] detected through ARMS-PCR and Sanger sequencing. Parental segregation showed that the father carried only the IVS1-5G &gt; C mutation and remained clinically transfusion independent, whereas both siblings carried IVS1–5G &gt;C and Hb Oslo mutations, exhibited a transfusion-dependent phenotype. These findings suggest that while each variant alone is typically associated with a mild or carrier phenotype in the heterozygous state, their co-inheritance was associated with a phenotypic shift from non–transfusion-dependent thalassemia (NTDT) to transfusion-dependent thalassemia (TDT) suggesting that co-inheritance of these mutation may aggravate the severity of beta thalassemia. This is the first report of such a genetic combination providing new insights into the complex genotype-phenotype correlation in β-thalassemia.These findings emphasize the importance of comprehensive molecular screening, genetic counseling and prenatal diagnosis in high-prevalence regions.</p><p></p>