Studies on science education show that students have many misunderstandings and misconceptions related to chemistry concepts. Many techniques have been used to determine those misconceptions such as multiple choice tests, interviews, drawing, etc. Two-tier diagnostic test is one of these techniques for using to determine misconceptions and their reasons. For this reason, in this study, it was aimed to determine middle school students' misconceptions related to the unit of "Structure and Properties of Matter" using a Two-Tier Diagnostic Test. The study was conducted with two hundred twenty five 7th grade students, who had already learned the unit of "Structure and Properties of Matter" and studied at three different middle schools in Istanbul. In this study "Structure and Properties of Matter Achievement Test", which was developed by the researchers and ensured validity and reliability, was used to collect data. The test was a two-tier diagnostic test that consists 32 items. Reliability coefficient was found to be 0.90. According to the results, it was found that students had high level of misconceptions about the topics "Stable Atom, Ion, Anion, Cation, Ionic Bond, Covalent Bond, Compound, Homogenous and Heterogeneous Mixtures, Concentrated and Dilute Solutions".