Introduction: Acceptance of a screening programme for thalassemia is important in managing the disease and its associated complications. The objective of this study was to determine the knowledge of thalassemia and factors associated with thalassemia screening refusal among parents of secondary school children. Methods: This cross-sectional study was carried out from May 2017 to October 2017 among parents of fourth form students in three schools in Besut, Terengganu, Malaysia. Parents who are able to read and understand Malay and consented to the study were required to answer a validated questionnaire on their knowledge regarding thalassemia. They were also asked the reason for their acceptance or refusal of the thalassemia screening of their children. Results: In total, 273 participants took part in the study. The mean thalassemia knowledge score was 11.8 out of a maximum score of 21. Low knowledge scores (adjusted odds ratio [adj OR] 0.87; 95% confidence interval [CI]: 0.79, 0.95; P = 0.002) and female sex (adj OR 2.60; 95% CI: 1.04, 6.53; P = 0.040) had significant associations with parental thalassemia screening refusal. The main reason for screening refusal was that parents perceived that their children were not at risk for the disease since they did not have a family member with thalassemia. Conclusion: The mean thalassemia knowledge score among parents remains unsatisfactory. A high knowledge score is important since it is associated with parental acceptance of thalassemia screening for their children.