Objective: To assess the attitude and willingness of medical students of the Faculty of Medicine, University of Jaffna, regarding gamete donation. Methods: An institutional-based descriptive cross-sectional study was conducted at the Faculty of Medicine, University of Jaffna, from September 2022 to May 2023 among undergraduate medical students who gave their voluntary participation. A self-administered questionnaire was used as a study instrument to collect data regarding their attitude and willingness toward gamete donation. Results: A total of 345 participants were recruited and their socio- demographic data revealed that 56.8% of the participants were female, 62.3% aged between 26 and 30 years, and 92.2% were unmarried. Many of them received information regarding gamete donations during their clinical appointments. Over half (67.8%) of them showed a negative attitude towards gamete donation. Regarding willingness, only 39.7% of participants had a positive approach for being a gamete donor; among them, 84.7% preferred anonymous donations. Religion and ethnicity had a significant influence on their attitudes and willingness. In addition, male was also found to be more willing to donate gametes. Conclusions: Most medical students have negative views about gamete donation. Imparting awareness and knowledge of assisted reproductive technology and gamete donation within medical students' sociocultural and ethical backgrounds might facilitate a change in attitude towards gamete donation amongst future medical practitioners.