α-Thalassemia (α-thal) is considered as the most common inherited hemoglobin disorder worldwide. The present study describes the first observation of a combination of rare α-chain variants, and β-globin gene cluster deletion. A 21-year-old woman with thalassemia trait, marked microcytosis, mild anemia, and normal range of Hb F was referred to Amirkola genetic center in the North of Iran for routine molecular test of thalassemia in the context of carrier detection and prevention of thalassemia major birth. Nucleotide sequencing revealed a novel compound heterozygosity status for two non-deletional mutations on , Hb O Indonesia (α116(GH4)Glu → Lys), and Hb Matsue-Oki (α75 (EF4) Asp → Asn), together with heterozygosity for the sicilian (δβ)-thal mutation. This finding highlights the necessity of deep molecular investigation of thalassemia in regions where thalassemia is abundant, and present highly heterogeneous population.