Background: Breast milk is the complete nutrition for the babies during first 6 months of life. It is very important to feed the baby with breast milk during that crucial period of growth. Unavailability of breast milk can put the baby in life threatening conditions. In order to resolve the problem and to improve the mortality rate of neonates, nurses should have up to date information regarding the breast milk and its storage. Aim of this study was to assess the knowledge of nurses regarding importance of human milk and milk banking.Methods: Quantitative, descriptive research approach was used to assess the knowledge of 150 purposively selected nurses working in obstetric, gynae and pediatric wards in selected hospitals of Punjab by using socio-demographic sheet and self-structured questionnaire. Both descriptive and inferential statistical methods were used for the analysis of data.Results: Study findings revealed that majority of nurses 66% have adequate knowledge regarding importance of human milk and milk banking. Knowledge of nurses regarding importance of human milk and milk banking is significantly associated with working department.Conclusions: Study concluded that knowledge of nurses regarding importance of human milk and milk banking is satisfactory. But for the further improvement in knowledge of nurse’s in-service programs should be provided to them after regular intervals of time. So that they perform their work efficiently.