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Abstract There is strong agreement in science teacher education of the importance of teachers' content knowledge for teaching (CKT), which includes their subject matter knowledge and their pedagogical content knowledge. However, there are limited instruments that can be easily administered and scored on a large scale to assess and study elementary science teachers' CKT. Such measures would support strategic monitoring of large groups of science teachers' CKT and the investigation of comparative questions about science teachers' CKT longitudinally across the professional continuum or across teacher education or professional development sites. To address this gap, this study focused on designing an automatically scorable summative assessment that can be used to measure preservice elementary teachers' (PSETs') CKT in one high‐leverage science content area: matter and its interactions. We conducted a field test of this CKT instrument with 822 PSETs from across the United States and used the response data to examine how this instrument functions as a potential tool for measuring PSETs' CKT in this science content area. Results suggest this instrument is reliable and can be used on large scale to support valid inferences about PSETs' CKT in this content area. In addition, the dimensionality analysis showed that all items measure a single construct of CKT about matter and its interactions, as participants did not show any differential performance by content topic or work of teaching science instructional tool categories. Implications for progressing the field's understanding of the nature of CKT and approaches to developing summative instruments to assess science teachers' CKT are discussed. 

Abstract Despite 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.