Iron deficiency (ID) and beta thalassemia trait (betaTT) are the most common causes of hypochromia and microcytosis. This study evaluates the reliability of some of the red blood cell (RBC) indices and the formulas used in the differentiation between betaTT and ID in a cohort of 458 children aged between 1.8 and 7.5 years (mean age 5.6+/-1.7 years) with mild hypochromic microcytic anemia. Within this group, 243 were confirmed with ID and 215 with betaTT. Red cell indices derived from automated red cell analyzers were used to evaluate the following discriminant indices and formulas: Mentzer Index (MI), Green and King Index (G&K), England and Fraser Index (E&F), RBC Distribution Width Index (RDWI), RBC distribution width (RDW) and RBC count. Sensitivity (SENS), specificity (SPEC), positive and negative prognostic value, efficiency (EFF) and Youden's Index (YI) were evaluated. For each index or formula Gauss curves were constructed. The highest SENS was obtained with RDWI (78.9%), while the highest SPEC and YI with E&F (99.1 and 64.2% respectively), the highest EFF (80.2%) with G&K. Gauss curves obtained from betaTT and ID children showed a different degree of overlap for each formula or index. In conclusion, none of RBC indices or formulas appears reliable to discriminate between betaTT and ID subjects.