Breastfeeding is the natural feeding. It is the ideal and uniquely superior food for infant. Nurses working for the care of new born should have the knowledge of storage and preservation of breast milk12. This study aimed to assess the knowledge and attitude of staff nurses working in NICU, PICU, Antenatal, postnatal and labour room of the selected hospitals of Gwalior city. A quantitative approach with non-experimental exploratory research design was adopted. 60 staff nurses were selected by non-probability convenient sampling technique. Data was collected using a structured questionnaire containing 40 multiple choice questions for knowledge and a rating scale with 20 items for attitude assessment and the data was analyzed using descriptive and inferential statistics. Study findings revealed that 15% staff nurses had poor knowledge, 66.66% had average knowledge and 18.37% had good knowledge. In case of attitude 68.33%, 3.33%, 28.33% have positive, negative and neutral attitude respectively among the staff nurses regarding storage of breast milk. A weak correlation (0.2) was found between knowledge and attitude. Religion had a significant relationship with knowledge whereas age and source of knowledge had a high degree of significance with attitude (at p <0.05 level). It was concluded that staff nurses had average knowledge with a positive attitude towards the storage of breast milk. An information booklet was developed and distributed to the staff nurses for further reference.