Background The Centers for Disease Control and Prevention (CDC) is a national public health protection agency in the United States. With the escalating impact of the COVID-19 pandemic on society in the United States and around the world, the CDC has become one of the focal points of public discussion. Objective This study aims to identify the topics and their overarching themes emerging from the public COVID-19-related discussion about the CDC on Twitter and to further provide insight into public's concerns, focus of attention, perception of the CDC's current performance, and expectations from the CDC. Methods Tweets were downloaded from a large-scale COVID-19 Twitter chatter data set from March 11, 2020, when the World Health Organization declared COVID-19 a pandemic, to August 14, 2020. We used R (The R Foundation) to clean the tweets and retain tweets that contained any of five specific keywords—cdc, CDC, centers for disease control and prevention, CDCgov, and cdcgov—while eliminating all 91 tweets posted by the CDC itself. The final data set included in the analysis consisted of 290,764 unique tweets from 152,314 different users. We used R to perform the latent Dirichlet allocation algorithm for topic modeling. Results The Twitter data generated 16 topics that the public linked to the CDC when they talked about COVID-19. Among the topics, the most discussed was COVID-19 death counts, accounting for 12.16% (n=35,347) of the total 290,764 tweets in the analysis, followed by general opinions about the credibility of the CDC and other authorities and the CDC's COVID-19 guidelines, with over 20,000 tweets for each. The 16 topics fell into four overarching themes: knowing the virus and the situation, policy and government actions, response guidelines, and general opinion about credibility. Conclusions Social media platforms, such as Twitter, provide valuable databases for public opinion. In a protracted pandemic, such as COVID-19, quickly and efficiently identifying the topics within the public discussion on Twitter would help public health agencies improve the next-round communication with the public.