Abstract Most Americans reject some or all parts of evolutionary theory, contrary to the scientific consensus. Americans’ attitudes toward evolution at least partially depend on their religious or political values, and prior work has argued that knowledge of the theory plays a negligible role. But there have been no systematic, large-scale attempts to measure the public’s knowledge of evolutionary theory, which means that claims about a lack of significant impact of evolution knowledge on evolution acceptance may be premature. Using a new demographically representative survey (N = 1100) that includes a detailed measure of evolution knowledge, we find that knowledge predicts level of acceptance, even after accounting for the effects of religion and politics. These results demonstrate that Americans’ views on evolution are significantly influenced by their knowledge about this theory and therefore might be amenable to change.