Background and Objective: Health literacy is a vital part of access and use of health interventions and well-being framework. However, there is a dearth of knowledge on health literacy levels of primary caregivers of children in Ghana. This study sought to investigate health literacy levels of mothers of children under the age of five years. Material and Methods: A community-based cross-sectional study was conducted among 1, 234 mothers who had children aged <5 years in Ejisu-Juaben and Kwabre-East, two randomly selected districts, in the Ashanti Region of Ghana from January 1 to March 30, 2021. A consecutive sampling technique was used to select the mothers to participate in the study. Data was collected using the Health Literacy Questionnaire (HLQ) and analyzed using Statistical Package for Social Sciences (SPSS) software version 20. Sample mean scores were presented in the form of a table. A paired-simple t-test was used to examine the significant levels of the differences in the mean health literacy scores of the mothers in the two districts. A multiple logistic regression analysis was then used to examine the relationships between the demographic variables of the respondents and health literacy. Results: On a nine-point health literacy scores, mothers scored above average on scale 1-5 (mean = 2. 61, ranges 2. 55-2. 68), while scale 5-9 had mean = 3. 39 (ranges 3. 33-3. 44). Scales 3 and 6, ‘, Actively managing my health’,(mean = 2. 68) and ‘, Ability to actively engage with healthcare providers’,(mean = 3. 44), had the highest scores while scales 1 and 7, ‘, Feeling understood and supported by healthcare providers’,(mean = 2. 55) and ‘, Navigating the healthcare system’,(mean = 3. 33), had the lowest scores. None of the HLQ scale items had a score below average. In the multiple logistic regression analysis, three variables had significant association with the mothers’,health literacy: age, educational level and speaking English at home. There was no statistically significant difference (p>0. 05) in the mean health literacy scores between the two studied districts. Conclusion: The mothers’,health literacy levels were above average. However, the results are not an indication of an outstanding performance. Thus, the country’, s healthcare professionals and policy makers still need to do a lot to ensure that mothers in the country attain the level of health literacy they need to improve their health, as well as that of their children.