α-Thalassemia (α-thal) is an inherited blood disorder with different clinical manifestations. Although genetic causes of anemia are identified routinely in the majority of α-thal cases, a pathogenic variant in a few cases remains undiagnosed. In this study, some reported regulatory mutations have been investigated in five unsolved α-thal carriers. α-Major regulatory element (α-MRE) haplotype analysis has also been performed in Iran for the first time. Four regions, including the HBA2 core promoter, the highly conserved sequence of hypersensitive-40 (HS-40), a region containing regulatory single nucleotide polymorphism (SNP) CR062116, and a region containing rs7203560, were screened for changes by Sanger sequencing in a total of five unsolved suspected α-thal carriers. The frequencies of α-MRE haplotypes B and C were also determined in control samples with normal hematological indices. No pathogenic variant was found in the investigated regions. Haplotype frequencies observed for B and C haplotypes fell into the range of frequencies observed in previous studies. The investigated genotypes in the control group were in the Hardy-Weinberg equilibrium. This study can provide evidence that there is no association between the B haplotype and microcytic hypochromic anemia. The cause of anemia remains a mystery in our unsolved cases, which demonstrates the need for further studies on the causes of hypochromic microcytic anemia in individuals with intact α- and β-globin genes without iron deficiency.