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Lesson observations and observation-based feedback are considered valuable in helping preservice teachers learn how to teach different subjects effectively, including science instruction. Observers’ content knowledge and their pedagogical content understanding presumably influence the quality of their observation focuses and the feedback they provide. This study examines the observation focuses and relevant feedback related to biology instruction by four groups of observers with different levels of content and pedagogical content knowledge for teaching biology, drawing on observation reports and interviews. The study found that the teachers’ content understanding alone may not determine the quality of their observation focuses and feedback. Instead, their pedagogical content understanding influences their observation focus and the quality of their feedback.