Objective: This study was conducted to determine perceptions regarding retention, application, and relevance of pre-clinical sciences in clinical training and practice, among final-year medical students and intern medical officers (IMOs) who graduated from the Rajarata University of Sri Lanka.Methods: A descriptive cross-sectional study was conducted using an online questionnaire, and the data were analyzed using descriptive statistics and t-test.Results: A total of 211; 110 final-year students (52.1%) and 101 IMOs (47.9%) responded. Perceived knowledge retention was highest in Physiology (5.73+1.81/10), followed by Anatomy (5.12+1.7/10) and Biochemistry (3.99+1.97/10). The majority reported higher retention of clinically relevant concepts (79.6%) and a gradual decline in retained knowledge during clinical years (75.8%). Content areas with the highest perceived clinical relevance were neuroanatomy, abdomen, pelvis and perineum, radiological anatomy, and head and neck in Anatomy, blood, gastrointestinal and hepatobiliary, and endocrine Biochemistry and physiology of cardiovascular, respiratory, endocrine, urinary, gastrointestinal and reproductive systems. Knowledge on biomolecules, metabolism, molecular genetics, histology, embryology and genetics were considered least clinically relevant. Pre-clinical sciences were perceived as fundamental for clinical training by the majority. They perceived that Anatomy and Physiology were applied more in diagnosis and patient management than Biochemistry. The majority expressed the need to revisit and re-evaluate Anatomy and Physiology during the clinical phase.Conclusion: A high level of relevance of pre-clinical sciences in many content areas was perceived by the participants. The study highlights the need for an early introduction to the clinical concepts within the preclinical curriculum and revisiting preclinical sciences during clinical training.