Microcytic hypochromic anemia is a common condition in clinical practice and alpha-thalassemia has to be considered as a differential diagnosis. Molecular diagnosis of alph-thalassemia is possible by polymerase chain reaction. The aim of this study was to evaluate the frequency of alpha-gene numbers in subjects with microcytosis. In total, 276 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] were studied. These include 125 with thalassemia trait, 48 with thalassemia major, 26 with sickle-cell thalassemia, 15 with E beta-thalassemia, 40 with iron-deficiency anemia, 8 with another hemolytic anemia, and 14 patients with no definite diagnosis. Genotyping for -alpha 3.7 deletion, alpha-4.2 deletion, Hb Constant Spring, and -triplications was done with polymerase chain reaction. The overall frequency of -alpha 3.7 deletion in 276 individuals is 12.7%. The calculated allele frequency for -thalassemia is 0.09. The subgroup analysis showed that co-inheritance of -deletion is more frequent with the sickle-cell mutation than in other groups. We were able to diagnose 1/3 of unexplained cases of microcytosis as -thalassemia carriers. The -gene mutation is quite common in the Indian subcontinent. Molecular genotyping of -thalassemia helps to diagnose unexplained microcytosis, and thus prevents unnecessary iron supplementation.