BACKGROUND Microcytic hypochromic anemia is a common condition in clinical practice, and alpha-thalassemia has to be considered as a differential diagnosis. OBJECTIVE 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. METHODS 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. METHODS Statistical analysis was carried out by SPSS 11.5 and an independent-sample t test. RESULTS 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). CONCLUSIONS 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.