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Low acceptance of evolution remains an obstacle to quality biology instruction. We develop and utilize a novel assessment which measures emotional experience in light of real-world evolution education scenarios. We presented 296 science teachers 4 pro-evolution and 8 anti-evolution scenarios and asked them to rate their levels of joy, anger, sadness, fear, disgust, shame, and guilt elicited by that scenario on an ordinal 5-point scale. We used exploratory factor analysis to extract the most important dimensions in the teachers' responses, Rasch analysis to explore the validity of the extracted subscales, and stepwise regression to find the most important factors driving emotional dispositions. We extracted 3 factors: (1) pro-evolution experience (positive emotions on pro-evolution and negative emotions on anti-evolution scenarios), (2) anti-evolution experience (negative emotions on pro-evolution and positive emotions on anti-evolution scenarios), and (2) feelings of regret over anti-evolution scenarios (shame and guilt on anti-evolution scenarios). Acceptance of evolution facts and a non-theistic religious orientation were positively related to pro-evolution experience. Anti-evolution experience was predicted by lack of microevolution acceptance and lack of teacher preparation. Feelings of regret around anti-evolution scenarios were driven by acceptance of evolution facts and lower levels of teacher preparation. This work advances our understanding of how teachers relate affectively to the theory of evolution and offers empirical insight into ways to improve dispositions about evolution.