Immunization is one of the most effective and cost-effective ways to protect children’s lives and futures. Knowledge of mothers on immunization plays a major role in the immunization of the children. This study was conducted to assess the knowledge of mothers on immunization of the children. This was a cross-sectional study, conducted among 120 mothers who were selected using the purposive sampling technique, in 2020 in Chennai, India. Mothers having children below the age of 5 years were included in the study. Data were collected by interviews at their homes using a pretested, predetermined, and structured questionnaire. Data is entered in Excel and analyzed in SPSS 20 using appropriate descriptive ( f, %, Mean, and SD) and inferential statistics ( t-test, ANOVA, and chi-square test). The mean knowledge score on immunization was 10.75/20. The majority of the mothers had moderately adequate knowledge (63.3%), followed by inadequate knowledge (30%), and only 6.7% of them had adequate knowledge. Regarding immunization coverage, the majority of the children were fully immunized in time (74.2%), followed by delayed immunization (15.8%) and only 10% were non-immunized. Mothers’ knowledge on immunization status was significantly associated with residence ( Living area, t= 2.13, p=0.035) and educational status of the mothers ( F= 0.0979, p= 0.379). Collected data is entered in Excel and analyzed in SPSS 20 using appropriate descriptive ( f, %, Mean, and SD) and inferential statistics ( t-test, ANOVA, and chi-square test). Educating mothers about immunization focusing on its benefits and cost-effectiveness using various strategies should be planned to increase the mothers’ knowledge on immunization and immunization coverage.