ABSTRACT Background: Recent outbreaks of Zika virus around the world led to increased discussions about this issue on social media platforms such as Twitter. These discussions may provide useful information about attitudes, knowledge, and behaviors of the population regarding issues that are important for public policy. Objective: We sought to identify the associations of the topics of discussions on Twitter and survey measures of Zika-related attitudes, knowledge, and behaviors, not solely based upon the volume of such discussions but by analyzing the content of conversations using probabilistic techniques. Methods: Using probabilistic topic modeling with US county and week as the unit of analysis, we analyzed the content of Twitter online communications to identify topics related to the reported attitudes, knowledge, and behaviors captured in a national representative survey (N=33,193) of the US adult population over 33 weeks. Results: Our analyses revealed topics related to “congress funding for Zika,” “microcephaly,” “Zika-related travel discussions,” “insect repellent,” “blood transfusion technology,” and “Zika in Miami” were associated with our survey measures of attitudes, knowledge, and behaviors observed over the period of the study. Conclusions: Our results demonstrated that it is possible to uncover topics of discussions from Twitter communications that are associated with the Zika-related attitudes, knowledge, and behaviors of populations over time. Social media data can be used as a complementary source of information alongside traditional data sources to gauge the patterns of attitudes, knowledge, and behaviors in a population.