Microcytic hypochromic anemia is a common condition in clinical practice, and alpha-thalassemia has to be considered as a differential diagnosis.This study was conducted to evaluate the frequency of alpha-gene, beta-gene and hemoglobin variant numbers in subjects with microcytic hypochromic anemia.Population-based case-control study in the Iranian population.A total of 340 subjects from southwest part of Iran were studied in the Research Center of Thalassemia and Hemoglobinopathies (RCTH), Iran. Genotyping for known alpha- and beta-gene mutations was done with gap-PCR and ARMS. In cases of some rare mutations, the genotyping was done with the help of other techniques such as RFLP and ARMS-PCR.Statistical analysis was carried out by SPSS 11.5 and an independent-sample t test.Out of the total 340 individuals, 325 individuals were evaluated to have microcytic hypochromic anemia based on initial hematological parameters such as MCV<80 fl; MCH<27 pg; the remaining 15 patients were diagnosed with no definite etiology. The overall frequency of -alpha3.7 deletion in 325 individuals was 20.3%. The most frequent mutations were IVS II-I, CD 36/37 and IVS I-110 with frequencies of 6.31%, 5.27% and 1.64%, respectively. Only, there was a significant difference between beta-thalassemia trait and beta-thalassemia major with regard to MCV (P<0.05) and MCH (P<0.05) indices, and also MCH index between beta-thalassemia trait and Hb variants (P<0.05).Molecular genotyping provides a rapid and reliable method for identification of common, rare and unknown alpha- and beta-gene mutations, which help to diagnose unexplained microcytosis and thus prevent unnecessary iron supplementation.