Some developing countries are currently introducing and implementing an electronic medical record system (EMRs) for improvement in healthcare delivery services. Availability of information and communication technology (ICT), technical skillful staff, and strong resistance to change by the health professionals impacted the successful adoption of EMRs. This study aimed to assess the ICT literacy, knowledge, and readiness for EMRs adoption among health professionals in a tertiary hospital, Myanmar. A cross-sectional study was conducted among 118 health professionals involving in a tertiary hospital at Nay Pyi Taw, Myanmar from February to April 2020 using a stratified sampling method. The data were collected through face-to-face interviews using a pretested structured questionnaire after getting informed consent. Data were analyzed by using SPSS version 23.0. Chi-square test, Fisher's exact test, and logistic regression analysis were performed to assess the associated factors of ICT literacy, knowledge, and overall readiness for EMRs adoption. The prevalence of high ICT literacy and knowledge on EMRs among health professionals were 20.3% and 24.6% respectively. The factors associated with ICT literacy were professional, education, duration of service, and reported English language skills. Duration of service was associated with knowledge on EMRs. The overall readiness was 54.2% (core readiness 59.3% and engagement readiness 61.9%), and postgraduate [Adjusted Odds Ratio (AOR): 7.32, 95% Confidence Interval (CI): 2.26-23.68] and knowledge on EMRs (AOR: 1.27, 95% CI: 1.13-1.43) were the factors associated with overall readiness for EMRs adoption. Expanding infrastructure and provision of ICT development training are crucial for the improvement of ICT literacy. EMRs training program enabling hands-on experience should be implemented for improvement of knowledge on EMRs. In general, the overall readiness for EMRs adoption was found to be moderate. Enhancing the establishment of comprehensive on-the-job training and contextualization of curriculum in EMRs training program are recommended to improve the health professionals' readiness for EMRs adoption.