University students' misconceptions have become an important issue to discuss as prior concepts brought by the students into the classroom can affect the acquisition of new information. The current study attempted to identify Biology students' misconceptions in Human Anatomy and Physiology courses through a three-tier diagnostic test. The participants of this study were 128 students from the Department of Biology Education who were currently enrolled in Human Anatomy and Physiology courses at the Institute of Teacher Training and Educational Sciences (STKIP) Pembangunan Indonesia in Makassar. A three-tier diagnostic test was used as the instrument to identify the number of participants who had developed misconceptions about the concepts introduced in the courses. The test contained 23 items of Certainty of Response Index (CRI) that, on average, achieved a validity score of 0.426 and a reliability score of 0.794. The research data were analyzed using descriptive statistics. The results of the test analysis showed that the majority (61.51%) of the students developed misconceptions in Human Anatomy and Physiology courses. In detail, there were several misconceptions in 57.81% skeletal system, 52.34% muscular system, 55.47% integumentary system, 51.04% nervous system, 54.69% endocrine system, 66.02% hemolymphatic system, 62.89% cardiovascular system, 68.75% respiratory system, 70.31% digestive system, 70.70% urinary system, and 71.88% reproductive system. Misconceptions were mostly developed on reproductive system sub-materials and less likely found in nervous system sub-materials. Given this information, it can be concluded that the misconceptions of Biology students from the Institute of Teacher Training and Educational Sciences (STKIP) Pembangunan Indonesia in Human Anatomy and Physiology courses are categorized as high. Therefore, there should be an effort to deal with this issue.