Over the past decades, a large body of research has examined students' magnitudes of evolution acceptance and related measurement issues resulting in questions concerning instruments' validity and operationalization. Until now, several studies investigated validity aspects of often-used evolution acceptance instruments and came to diverging conclusions about instruments' scores comparability. Within the last years, religious identity was identified as a significant predictor for magnitudes of evolution acceptance. However, religious identity can also point to validity issues if aspects of the content under investigation are interpreted differently based on specific religious identity. Additionally, self-identified creationists could serve as a source of validity evidence due to the assumption that creationists should score lower on an evolution acceptance instrument than groups with more scientifically adequate views. Thus, we aim to provide additional validity evidence for often-used evolution acceptance instruments within a European context (i.e., Germany) by comparing two groups of particular interest for research in science education: preservice biology teachers and self-identified creationists. Exploring evidence based on (1) internal structure, (2) relationships with other variables, and (3) test content provides arguments for test interpretations' validity. A total of 286 persons (206 preservice biology teachers and 81 self-identified creationists) participated in a survey comprised of six often-used evolution acceptance measures (i.e., MATE, GAENE, I-SEA, ATEVO, Gallup question, and 100 point questions). Overall, our findings indicate that the six applied instruments demonstrate differences concerning their evidence for sound interpretation. In particular, the DIF and dimensionality analyses lead to the assumption that the GAENE may be multidimensional other than theoretically expected. In general, the multi-item measures were stronger related to each other than to the general public polls. Using an instrument that differentiates between microevolution and macroevolution is advisable for creationist samples or samples with a remarkable share of creationists' views.