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According to the Thalassemia Federation of Pakistan, the mostly inherited disorder in Pakistan is β-thalassemia, which is characterized by a deficient, abnormal, or lack of β-globin chain synthesis and has a prevalence of 6%. The only method of controlling and preventing β-thalassemia is to increase awareness among students. This was an observational study using a random sampling technique. The Dow-Thalassemia awareness program recruited 915 medical students from the Dow Medical College (DMC) and Sindh Medical College (SMC) to voluntarily donate blood samples, which were analyzed by the naked eye single tube red cell osmotic fragility test (NESTROFT) and complete blood count and results were confirmed by high-performance liquid chromatography and analyzed using the NESTROFT. The samples were collected in 2012-2013. A total of 915 samples, out of these 390 samples, 390/915 (42.6%) samples were positive and complete blood count found 282 (72.3%) were positive for iron deficiency anemia. The remaining 108/390 (27.6%) were confirmed by high-performance liquid chromatography. Only 2.4 % subjects were positive for the β-thalassemia trait. Of 915 students, 57.4% of students were healthy, 39.2% had iron deficiency anemia, and 2.4% were carriers of the β-thalassemia trait. The overall prevalence of β-thalassemia was 38/915 (4.1%), which was lower than observed in previous studies. This study also demonstrated the NESTROFT can be used as a primary method of screening out healthy individuals, where approximately 50% require further screening for β-thalassemia