Iron deficiency anaemia is one of the main causes of anemia during pregnancy as it is associated with increased maternal and perinatal mortality and morbidity. A descriptive and cross-sectional study was carried out to assess the knowledge and practice of mothers regarding the prevention of anemia during pregnancy among mothers who delivered in Tribhuvan University Teaching Hospital (TUTH). Convenient purposive sampling technique was adopted and mothers were included in the study who delivered in TUTH during four weeks period of data collection. They were interviewed by administering semi structured questionnaire. Chi-square test was applied to assess the association between variables. The test result shows that there was significant association in level of knowledge to the educational status regarding prevention of anemia during pregnancy (p=0.002). Furthermore, the study also revealed that there was significant association between frequencies of antenatal care (ANC) visit to the level of knowledge (p=0.007) as well as level of practice (p=0.043) of mothers about pre­vention of anemia in pregnancy. From the result of the study, it is concluded that the majority of mothers had not adequate knowledge and poor practice regarding prevention of anemia during pregnancy.