Background: Thalassemia is one of the most common genetic blood disorders in Asia. Consanguineous marriages and avoiding pre-marital and antenatal screening are common in Pakistan due to psychosocial, cultural, and religious factors. Few studies have investigated the beliefs regarding thalassemia, especially in a developing country. The aim of this study was to assess the knowledge, beliefs, and practices regarding thalassemia in an urban population.Method: A cross-sectional descriptive study was conducted in the urban areas of Karachi, Pakistan over a period of six months during March 2016 through August 2016. Participants selected by representative sampling were interviewed face-to-face using a pre-designed, pre-tested questionnaire. The questionnaire was divided into four parts. The first part inquired about general demographic variables, while the second part assessed knowledge of the participant regarding thalassemia. The third and fourth parts were concerning the beliefs and practices regarding thalassemia. Data were entered and analyzed using the Statistical Package for Social Sciences (SPSS) Statistics, v. 24.0 (IBM SPSS Statistics, Armonk, NY).Results: Only 53% (n = 720) of the respondents had heard about thalassemia. The mean knowledge score was 5.8. The total possible score ranged between 0 - 12 with the higher scores indicating better knowledge. About three-quarters (70%) of the sample did not know that an individual can be a carrier of thalassemia. Less than half (38%) of the participants viewed premarital screening for thalassemia as necessary, with only 10% agreeing that thalassemia carriers should not marry. There was no pre-marriage counseling done in 98% of the families. Conclusion: Our study highlights the prevalence of numerous myths and a low level of knowledge regarding thalassemia in an urban population of Pakistan.