Background: Thalassemia, an inherited blood disorder represents a significant burden for many countries across the globe and the knowledge related to this illness is very limited. The preventive practices, among people especially from developing countries. In Pakistan, Thalassemia is a serious health problem bearing 5 to 8% frequency of beta-Thalassemia gene without discrimination of ethnic groups. Objective: To assess the awareness level regarding Thalassemia in general population of Rahim Yar Khan, a district of Southern Punjab, Pakistan. Methodology: Study Design: Cross sectional study. A total of 400 adults of 18 years and above age, were selected randomly, from the households of the Rahim Yar Khan City and one Rural Union council. Out of 400 subjects, only 131, reported that they had heard the name ”Thalassemia”, hence for analysis of awareness, the data of only these 131 was used. A structured questionnaire was designed which included information on; gender, age and 24 questions about awareness of Thalassemia disease. Level of Awareness was ascertained by response of these 24 questions and was categorized as poor, average and good. The persons answering less than 12 questions correctly were considered to have poor knowledge, answers of 13-18 questions were labeled as possessing average knowledge and the persons were included in category of having good knowledge who answered more than 18 questions correctly. Data was analyzed by using SPSS 20. Results: Out of 131 study subjects , who reported to have ever heard of word Thalassemia, level of awareness about Thalassemia was found to be “Good” among 7 (5.4%) persons, 27 (20.6%) persons have an average knowledge about Thalassemia and a large number of people 97 (74%) have poor knowledge about Thalassemia disease. Conclusion: Public knowledge regarding Thalassemia was found to be poor among general population of Rahim Yar Khan, Pakistan.