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Background: Iatrogenic and nosocomial infections are a serious threat to a healthcare setting especially during a pandemic. Hand hygiene among the health-care workers stands out to be a pivotal preventive measure. Practical application of hand hygiene measures during third wave of COVID-19 pandemic primarily depend upon the current knowledge among the health care workers. Methods: A cross-sectional study design based on validated WHO questionnaire for hand hygiene among the health care workers was conducted during March till May2021 in the settings of Shalamar and Central Park hospital and medical colleges. Results: The sample consisted of 271 participants with 110 males and 161 females, with the mean age of 24.72+ 4.174 years. The sample consisted of 36.2% of medical students, 49.1% of doctors and 14.8% of paramedical staff. Nearly 50.6% of the individuals claim to receive formal training. 47.6% believe that “Health-care workers’ hands when not clean” are the main route of cross-transmission. Nearly 57.2% believed that the hospital environment (surfaces)the most frequent source of germs. Comparison of hand-rubbing and washing revealed that major percentage believe hand rub to be rapid however less effective causing dryness. A majority believe that hand hygiene before touching the patient, immediately after exposure to body fluids or immediate surroundings of patients prevents transmission of germs to the patient and vice versa afterwards prevents transmission to HCW. Conclusion: A large proportion of the sample has considerable knowledge regarding essential hand hygiene in a health care setting. However more frequent training sessions should be conducted to improve it further. Key words: Hand hygiene, Health-care workers, Knowledge