AbstractDespite the importance of developing elementary science teachers' content knowledge for teaching (CKT), there are limited assessments that have been designed to measure the full breadth of their CKT at scale. Our overall research project addressed this gap by developing an online assessment to measure elementary preservice teachers' CKT about matter and its interactions. This study, which was part of our larger project, reports on findings from one component of the item development process examining the construct validity of 118 different CKT about matter assessment items. In this study, 86 elementary teachers participated in cognitive interviews to examine: (a) the knowledge and reasoning they used when responding to these CKT about matter assessment items and (b) the nature of the content challenges and the content teaching challenges they encountered. Findings showed that over 80% of participant interview responses indicated that the CKT about matter items functioned as hypothesized, providing evidence to support future use of these items on a large‐scale assessment and in studies of science teachers' CKT. When responding to the items, participants showed evidence of four main challenges with the science content: (a) using scientific concepts to reason about science tasks, (b) using adequate evidence to reason about science phenomenon, (c) drawing upon examples of scientific phenomena, and (d) drawing upon science vocabulary. Findings also showed that participants experienced challenges regarding the following content teaching aspects when responding to these items: (a) connecting to key scientific concepts involved in the work of teaching science, (b) attending to instructional goal(s), and (c) recognizing features of grade‐level appropriateness. Implications for using CKT items as part of large‐scale science assessment systems and identifying areas to target in elementary science teachers' CKT development are addressed.