We developed and validated the Conceptual Assessment of Natural Selection (CANS), a multiple-choice test designed to assess how well college students understand the central principles of natural selection. The expert panel that reviewed the CANS concluded its questions were relevant to natural selection and generally did a good job sampling the specific concepts they were intended to assess. Student interviews confirmed questions on the CANS provided accurate reflections of how students think about natural selection. And, finally, statistical analysis of student responses using item response theory showed that the CANS did a very good job of estimating how well students understood natural selection. The empirical reliability of the CANS was substantially higher than the Force Concept Inventory, a highly regarded test in physics that has a similar purpose.