<p>The purpose of this study is twofold: (1) to develop and validate a five-tier diagnostic instrument for respiration and photosynthesis; (2) to uncover senior high school students' genuine misconceptions in Mainland China and explore the characteristics of their sources and formation mechanisms. To achieve these aims, a five-tier diagnostic instrument was developed and administered to 291 students across three senior high schools located in three official geographical regions of China. Rasch models were constructed, demonstrating the high reliability and validity of the instrument through the overall reliability, unidimensionality, item-person interaction, and goodness-of-fit. The prevalence of seven misconceptions exceeds 10%, with five characterized by mean confidence ranging from 3.5 to 4.0, identified as genuine misconceptions of moderate strength. Notably, four of these have not been previously reported in the literature. The research revealed that students harbored the same genuine misconceptions, which were obtained from either a single source or compound sources. Student interviews revealed that these critical factors, stemming from individual sources such as ineffective teaching methods, inaccuracies in textbook content and organization, and students' incorrect inferences, each independently contributes to the formation of genuine misconceptions. Additionally, the interaction between compound sources is also highlighted as a pivotal factor in the formation mechanisms of genuine misconceptions.</p>