Despite pediatric populations representing a smaller proportion of COVID-19 cases and having a less severe prognosis, those belonging to racial and ethnic minority groups are at an increased risk of developing more severe COVID-19-related outcomes. Vaccine coverage is crucial to pandemic mitigation efforts, yet since the start of the COVID-19 pandemic, vaccine hesitancy has increased and routine pediatric immunizations have decreased. Limited research exists on how vaccine hesitancy may contribute to low pediatric COVID-19 vaccine uptake among racial and ethnic minority populations. This study aimed to characterize COVID-19 vaccine-related discussion and sentiment among Twitter users, particularly among racial and ethnic minority users. We used the Twitter application programming interface to collect tweets and replies. Tweets were selected by filtering for keywords associated with COVID-19 vaccines and pediatric-related terms. From this corpus of tweets, we used the Biterm Topic Model to output topics and examined the top 200 retweeted tweets that were coded for pediatric COVID-19 vaccine relevance. Relevant tweets were analyzed using an inductive coding approach to characterize pediatric COVID-19 vaccine-related themes. Replies to relevant tweets were collected and coded. User metadata were assessed for self-reporting of race or ethnic group affiliation and verified account status. A total of 863,007 tweets were collected from October 2020 to October 2021. After outputting Biterm Topic Model topics and reviewing the 200 most retweeted tweets, 208,666 tweets and 3905 replies were identified as being pediatric COVID-19 vaccine related. The majority (150,262/208,666, 72.01%) of tweets expressed vaccine-related concerns. Among tweets discussing vaccine confidence, user replies expressing agreement were significantly outweighed by those expressing disagreement (1016/3106, 32.71% vs 2090/3106, 67.29%; P<.001). The main themes identified in the Twitter interactions were conversations regarding vaccine-related concerns including adverse side effects, concerns that the vaccine is experimental or needs more testing and should not be tested on pediatric populations, the perception that the vaccine is unnecessary given the perceived low risk of pediatric infection, and conversations associated with vaccine-related confidence (ie, the vaccine is protective). Among signal tweets and replies, we identified 418 users who self-identified as a racial minority individual and 40 who self-identified as an ethnic minority individual. Among the subcodes identified in this study, the vaccine being protective was the most discussed topic by racial and ethnic minority groups (305/444, 68.7%). Vaccine-related concerns can have negative consequences on vaccine uptake and participation in vaccine-related clinical trials. This can impact the uptake and development of safe and effective vaccines, especially among racial and ethnic minority populations.