Objective: Iron deficiency anemia (IDA) and beta-thalassemia trait ( -TT) are the most common forms of microcytic anemia. This study was conducted to compare the validity of various discrimination indices in differentiating β-TT from IDA by calculating their sensitivity, specificity and Youden's index.Methods: Totally 323 subjects (173 children and 150 adults) with microcytic anemia were involved in this study. We calculated 10 discrimination indices in all patients with IDA and β-TT. We divided the patients into two different groups as younger or older than 10 years. Results: None of the indices showed sensitivity and specificity of 100% in the patients older than 10 years, and in the patients younger than 10 years, only Shine & Lal index showed sensitivity close to 90% and specificity of 100%. The most accurate discriminative index for patients younger than 10 years was Shine & Lal and for those older than 10 years it was RDW index. According to Youden's index, Shine & Lal and RBC count showed the greatest diagnostic value in patients younger than 10 years and RDW and RBC count indices in those older than 10 years. Conclusion: None of the indices was completely sensitive and specific in differentiation between β-TT and IDA. Mean and median mean cell Hb density (MCHD) were very close to normal values in both IDA and β-TT patients, but in the case of mean density of Hb/liter (MDHL), we found that the mean and median were significantly higher than normal values in β-TT and lower than normal values in IDA patients. In our study, Youden's index of RBC and Shine & Lal were the highest and most reliable indices in differentiating β-TT from IDA in the patients younger than 10 years. For patients older than 10 years, the most reliable discrimination indices were RBC and RDW.