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This study examines the nature of preservice elementary teachers’ content knowledge for teaching (CKT) in one science content area: matter and its interactions. In this study, we developed and conducted a field test of a 60-item assessment with items aligned to both an instructional tool in the ‘Work of Teaching Science’ framework and one of five sub-content categories of matter. Findings from the field test analysis showed well-functioning items, high test reliability, and moderate relationships with external measures of similar but distinct constructs. Results also showed unidimensionality, suggesting that items assess the single construct of CKT about matter. This CKT science assessment can be used to measure science teachers’ CKT across contexts and over time in efficient and scalable ways.