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The pathways and transformation of matter and energy (PTEM) is a central organizing principle for systems-level thinking in biology, yet students face many challenges with this topic. In this study, we use the Movement of Matter in Organisms instrument (MOMO) to measure the magnitude and structure of students’ PTEM knowledge across biological phenomena using measures derived from Rasch modeling. We documented significant learning gains and coherence change across taxonomic contexts from pre- to post-test, but disparities emerged based on several student demographic characteristics following instruction. These findings offer insights about the potential of the MOMO to measure progress toward PTEM proficiency through principled use of phenomena and show how aligning assessment design with student cognition can support measurement across disciplines.