Alpha-thalassemia is a common inherited hemoglobin disorder caused by deletions or point mutations in the HBA1 and/or HBA2 genes, leading to reduced or absent synthesis of alpha-globin chains. Although deletional mutations are predominant, non-deletional mutations can also contribute to the clinical spectrum of the disease, with varying severity. This study aimed to characterize two novel mutations in the HBA1 gene, a 230 bp microdeletion and a missense mutation (NP_000549.1:p.Ala29Pro), identified in two unrelated Tunisian families. Additionally, we assessed the clinical, hematological, molecular, and in silico structural impact of these mutations. Peripheral blood samples were collected from affected individuals and family members. Hematological parameters were measured using automated cell counters, and hemoglobin fractions were analyzed by high-performance liquid chromatography (HPLC). Genomic DNA was extracted, and common α-thalassemia deletions were screened via gap-PCR. Negative cases underwent sequencing of HBA1 and HBA2 exons. The novel deletion was confirmed by gap-PCR, gel purification, and sequencing. The pathogenicity of the missense mutation was assessed using in silico tools (PolyPhen-2, SIFT, MutationTaster), and structural modeling was performed using Swiss-PdbViewer and DynaMut to evaluate protein stability and flexibility. In Family A, a 4-year-old proband exhibited mild anemia, microcytosis, hypochromia, and low ferritin levels unresponsive to iron supplementation. A novel heterozygous 230 bp deletion (NC_000016.10:g.176695_176925del), named Hemoglobin Ariana, was identified in the HBA1 gene, affecting the 5'UTR, exon 1, and part of intron 1. In Family B, a 4-month-old proband presented with co-inheritance of α-thalassemia and Hemoglobin C. Sequencing revealed a novel missense mutation (NM_000558.5:c.85G > C, NP_000549.1:p.Ala29Pro), designated Hemoglobin Tozeur. In silico analysis predicted the mutation as deleterious, with structural modeling indicating destabilization of the α-globin protein, reduced stability, and altered flexibility near the heme-binding region. This study identifies two novel HBA1 mutations associated with α-thalassemia in Tunisia. The combined clinical, molecular, and structural analyses highlight the importance of comprehensive genetic screening and in silico modeling for accurate diagnosis and improved management of thalassemia cases.