New parameters correlated with the hemoglobin content in reticulocytes (RET-Y) and in red blood cells (RBC-Y) have been suggested as helpful in diagnosing iron deficiency anemia. We have studied RET-Y and RBC-Y indices in two groups of patients with microcytosis to verify if these parameters could be used to differentiate iron deficiency anemia from β-thalassemia minor. Blood samples from 33 iron-deficient patients, 25 β-thalassemic minor patients and 50 normal individuals were analyzed on a Sysmex XE-2100 instrument. A significant difference was observed in reticulocyte counting and immature reticulocyte fraction between iron deficiency anemia and β-thalassemia minor groups, but not in RBC-X and RET-Y parameters. Reticulocyte counting was higher in β-thalassemia minor and the immature reticulocyte fraction was higher in severe iron deficiency anemia. The ratio RET-Y/mean cell volume was tested and was significantly different when β-thalassemia minor was compared with mild and severe iron deficiency anemia, and showed better performance than the Mentzer ratio and the Green and King function. A great overlap of RET-Y and RBC-Y individual values was observed in both groups of microcytic anemias; we conclude that these new indices may be used with caution as indicative of iron deficiency, mainly in populations where β-thalassemia minor is frequent.