The aim of this study is to examine the "Knowledge of Student Understanding (KSU)” of pre-service science teachers on "Heat and Temperature" topics. The sample of the research consists of 268 pre-service teachers studying at the 4th grade in the science teaching program of five different universities in Turkey. This research is a case study. The data of the study are collected with "Student Thinking Comprehension Test” and "Interview Form". "Scoring Rubrics for Knowledge of Student Thinking" and "Rubric for Analysis of Interviews" are used for the analysis of data. The data of the research are analyzed descriptively on the basis of these rubrics. The results of the study reveal that the vast majority of pre-service science teachers cannot effectively diagnose the student’s inaccurate knowledge that emerges in solving specific problems in “Heat and Temperature” topics. In addition, it is shown that the majority of pre-service science teachers have a little knowledge regarding the difficulties and limitations of students while learning "Heat and Temperature" subjects. According to these results, the KSU of the pre-service science teachers in "Heat and Temperature" topics is insufficient in the process of starting their profession.