The Reaction Coordinate Diagram Inventory (RCDI) has been developed to measure student thinking and confidence with reaction coordinate diagrams (RCDs) and their correspondence to reaction mechanisms. The RCDI was designed using a sequential mixed-methods protocol, such that the questions and distractors in the instrument were generated based on an analysis of qualitative semi-structured interviews in which general chemistry and organic chemistry students were asked to describe the concepts embedded in the surface features of RCDs and to use the mechanistic information encoded in RCDs. The RCDI was administered to second-semester general chemistry students (n = 443), second-semester organic chemistry students (n = 227), and second-semester physical/biophysical chemistry students (n = 45). Descriptive statistics and item function are presented for each of the three samples. Qualitative and quantitative evidence for the validity and reliability of the data generated by the RCDI are also presented. Major categories of misconceptions assessed by the RCDI and data demonstrating the prevalence of these misconceptions for all three groups of students are presented.