Background: Although pollinators play an integral role in human well-being, their continued global decline reflects the need to provide and evaluate general pollinator knowledge to promote their conservation. Enhancing learners' understanding of the complexity inherent in pollination systems within the science classroom may help them make more informed decisions regarding pollinator conservation actions. By measuring conceptual understanding of pollination systems, science educators can identify learners' knowledge needs and inform their teaching in science classrooms. Based on previously developed theoretical frameworks describing pollination systems knowledge, we created and evaluated a new instrument to assess pollination systems and conservation actions knowledge. The Pollination Systems Knowledge Assessment (PSKA) is a multiple-true-false instrument containing 18 question stems and 70 accompanying T-F items encompassing three organizational components of pollination knowledge regarding (1) plant structures, (2) pollinator structures and behaviors, and (3) pollination systems function and pollinator conservation. Results: We refined the PSKA based on expert discussions, think-aloud interviews, and pilot testing before and after presenting a wild pollinator conservation unit within a postsecondary science literacy course. The PSKA elucidated learners' misconceptions and revealed discriminating items from the three organizational components of pollination systems knowledge. Conclusions: The PSKA may aid educators in exploring learners' conceptual understanding, identifying areas of misconceptions, and refining educational programming aimed at improving learners' pollination systems knowledge.