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A mandatory National Premarital Thalassemia Screening Program was implemented in Jordan in 2004. This cross-sectional study aimed to assess the knowledge and attitudes of university students in North Jordan toward this program. Data was collected from 542 students from four universities (two public and two private universities) located in North Jordan, using a structured questionnaire. Results of t-test and analysis of variance (ANOVA) showed that while respondents had adequate knowledge of and positive attitudes toward the premarital screening program, there was still a lack of knowledge about the disease itself. Nearly half the respondents were under the impression that β-thalassemia (β-thal) is a disease that can be treated simply. One-third of the respondents believed that if both partners were carriers of β-thal they should proceed with marriage. Negative attitude was revealed when many respondents believed that diagnosing a family member as a carrier affects other family members' future marriage opportunities. Significant associations were detected between the knowledge scores and gender, urban/rural residence, and the university where the students were enrolled. Students in private universities showed significantly lower attitude scores. Consideration of prenatal diagnostic services as part of a β-thal prevention program is necessary. It would also be helpful to include information about β-thal as a preventable inherited illness with a severe debilitating impact on the family in the high school curriculum. There is also a need for social marketing of the program.