Evolution is widely regarded as biology's unifying theme, yet rates of rejection of evolutionary science remain high. Anecdotal evidence suggests that cognitive dissonance leading to an emotional response is a barrier to learning about and accepting evolution. We explored the hypothesis that students whose worldviews may be inconsistent with the acceptance of evolution generate detectable emotional responses in the form of physiological changes when exposed to evolutionary themes. Physiological data (respiratory rate, galvanic skin response, and heart rate) were collected from participants while they were asked “yes/no” questions, some of which referenced evolution. Questions were of three categories: relevant, irrelevant, and control. Authenticity of response rates to relevant questions such as “Do you believe in evolution?” were verified using visual inspection to compare degree of response rates with control questions, such as “Have you ever cheated on a test?” Our results support our hypothesis. Of the 33 participants included in our study, a majority of them produced detectable physiological changes indicating emotional responses when asked questions referencing evolution. The highest response rate (79%) was generated by the question “Do you believe in evolution?” The implications of an emotional response in students when presented with instruction in evolutionary theory are discussed.