Despite the importance of evolution to understand biology, there is significant evidence that many biology teachers have difficulties to successfully teach this topic. The purpose of this study is to describe procedures by which a paper-and-pencil instrument to assess teachers’ pedagogical content knowledge for evolution (PCKevo) was developed and validated. The instrument was created to measure the components proposed in the model of Magnusson et al. (1999), except for the orientations. The creation of the questionnaire was carried out through eleven steps: (1) conceptualization, (2–4) creation of items and rubric reviewed by experts (focus on content and construct validity), (5–7) piloting a 32-item questionnaire to 10 biology teachers (focus on face and construct validity and reliability), (8–10) piloting a 16-item questionnaire to 61 biology teachers (focus on discriminant and construct validity and reliability), (11) application of a different instrument (CoRe interview) to four teachers (focus on criterion validity). The final version of the questionnaire (which includes three PCK components) showed in a Rasch analysis for the reliability of the items (α = 0.90) and persons (α = 0.81) adequate values. When applying the final version of the questionnaire, there is also evidence of discriminant validity (differences between two groups of teachers with or without professional development in evolution education). The contributions of the PCKevo instrument for research topics and evolution teaching are discussed.