Pakistan has a high prevalence of β-thalassemia (β-thal) but lacks a screening program for its prevention. This questionnaire-based cross-sectional study was conducted in six randomly chosen non medical universities to assess the students' knowledge of β-thal and premarital screening, and their attitude towards such a program. Comparison was made between the respondents' attitude towards premarital screening before and after providing them some information regarding the disease. Only 54.5% (207) of 380 students had heard of β-thal, with a mean knowledge score of 13.0 ± 4.4 out of 27 questions. Most respondents were aware of the concept of premarital screening. Out of 207 students, 60.4% wanted to know if they were carriers, 69.1% wanted to know their spouse's carrier status and 59.4% wanted premarital screening to be made mandatory in Pakistan. These figures increased to 72.5, 78.3 and 67.6%, respectively after provision of written information (p values: 0.03, 0.02, and 0.01, respectively). The positive attitude towards premarital screening with low background knowledge of the disease highlights the need of a mass awareness campaign and subsequent implementation of a premarital screening program.