Evolution is controversial among students and religiosity, religious affiliation, understanding of evolution, and demographics are predictors of evolution acceptance. However, quantitative research has not explored the unique impact of student perceived conflict between their religion and evolution as a major factor influencing evolution acceptance. We developed an instrument with validity evidence called "Perceived Conflict between Evolution and Religion" (PCoRE). Using this measure, we find that, among students in 26 biology courses in 11 states, adding student perceived conflict between their religion and evolution to linear mixed models more than doubled the capacity of the models to predict evolution acceptance compared with models that only included religiosity, religious affiliation, understanding of evolution, and demographics. Student perceived conflict between evolution and their religion was the strongest predictor of evolution acceptance among all variables and mediated the impact of religiosity on evolution acceptance. These results build upon prior literature that suggests that reducing perceived conflict between students' religious beliefs and evolution can help raise evolution acceptance levels. Further, these results indicate that including measures of perceived conflict between religion and evolution in evolution acceptance studies in the future is important.