Background: The aim of this study was to estimate the prevalence of the beta thalassemia trait (BTT) and differentiate it from iron-deficiency anemia (IDA) among blood donors. Methods: A total of 1,000 samples from blood donors were subjected to complete hemogram with red blood cell indices. Further, Mentzer index (MI) was calculated for samples with mean corpuscular volume (MCV) below 80 fL and mean corpuscular hemoglobin (MCH) below 27 pg. Samples with Mentzer index ＜12 were subjected to naked-eye single-tube red cell osmotic fragility test (NESTROFT) followed by hemoglobin electrophoresis in positive cases. Serum ferritin was assessed in NESTROFT-negative cases. Results: The prevalence of BTT among blood donors was 3.7% and that of microcytosis among donors was 8.6%. The prevalence of BTT among microcytic donors was 41.8% while that among those with IDA was 11.6%. A value of MI ＜13 was highly sensitive in the diagnosis of BTT. MI ＞13 was found to have both high specificity and high sensitivity for diagnosing IDA. Conclusion A moderately high prevalence of BTT was observed among blood donors. Presently, no screening program is mandatory for screening of BTT among blood donors. Indices like MCV, MCH, and Mentzer Index were thus found to be effective to screen for BTT and IDA among blood donors, and NESTROFT was a cost-effective mass screening method to differentiate BTT and IDA.