Background Online information on COVID-19 vaccination may influence people’s perception and willingness to be vaccinated. Official websites of vaccination programs have not been systematically assessed before. Objective This study aims to assess and compare the readability and content quality of web-based information on COVID-19 vaccination posted on official/governmental websites. Furthermore, the relationship between evaluated website parameters and country vaccination rates were calculated. Methods By referring to an open data set hosted at Our World in Data, the 58 countries/regions with the highest total vaccination count as of July 8, 2021, were identified. Together with the websites from the World Health Organization and European Union, a total of 60 vaccination campaign websites were targeted. The “frequently asked questions” or “questions and answers” section of the websites were evaluated in terms of readability (Flesch Reading Ease score and Flesch-Kincaid Grade Level), quality (Health On the Net Foundation code [HONcode] certification and Quality Evaluation Scoring Tool), and content stating vaccination duration of protection and potential side effects. Results In terms of readability, the Flesch Reading Ease score of the vaccination frequently asked questions websites ranged between 11.2 and 69.5, with a mean of 40.9 (SD 13.2). Meanwhile, the Flesch-Kincaid Grade Level ranged between 6.5 and 17.6, with a mean of 12.1 (SD 2.8). In terms of quality, only 2 websites were HONcode certified, and the Quality Evaluation Scoring Tool score of the websites ranged between 7 and 20, with a mean of 15.3 (SD 3.1). Half of the websites (25/50) did not present a publication date or date of the last update. Regarding the duration of protection offered by the vaccines, 46% (23/50) of the websites stated that they do not know, and another 40% (20/50) did not address it. Five side effects of the vaccinations were most frequently mentioned, namely, fever/chill (41/50, 82%), various injection site discomfort events (eg, swelling, redness, or pain; 39/50, 78%), headache (36/50, 72%), fatigue (33/50, 66%), and muscle/joint pain (31/50, 62%). Conclusions In general, the content quality of most of the evaluated websites was good, but HONcode certification should be considered, content should be written in a more readable manner, and a publication date or date of the last update should be presented.