Background The diagnostic criterion for beta thalassemia trait (BTT) is elevated Hb-A 2 levels. Iron deficiency anemia (IDA) reduces the synthesis of Hb-A 2 , resulting in reduced Hb-A 2 levels, so patients with co-pathological conditions BTT with IDA, may have a normal level of Hb-A 2 . Many socio-economic factors like unawareness, poor diagnostic facilities, and cost of molecular diagnosis (for screening purposes) result in interpretation of these subjects as normal. Methods Venous blood samples from 200 unmarried females having a family history of thalassemia were collected, and basic hematological parameters, hemoglobin electrophoresis, and molecular analysis for beta thalassemia were done. Patients with IDA and patients with co-pathological conditions BTT and IDA were treated with oral iron. These subjects were then followed for a period of 20 weeks. Results Of the 200 females, 34 were found to be anemic. Hemoglobin electrophoresis identified 16 of these patients as BTT. Molecular analysis of all patients confirmed this diagnosis, but identified 8 additional patients with BTT. Eight patients that were not detected with hemoglobin electrophoresis were found to have co-pathology of BTT with IDA. Conclusion Patients with the co-pathological condition BTT with IDA may be interpreted as being normal, as they have normal Hb-A 2 levels. These misdiagnosed subjects when marry with BTT have the potential to produce beta thalassemia major in offspring. This is one of the factors playing a major role in the propagation of beta thalassemia gene in Pakistani population, and become a serious hindrance for the thalassemia prevention program in Pakistan.