The purpose of this study was to explore Pre-service Elementary Science Teachers' (PSTs) conceptual understanding of Particulate Nature of Matter (PNM) through a three-tier diagnostic test. Participants were 215 PSTs from Turkey. Data consisted of participants' responses to the Particulate Nature of Matter Test (PNMT). The PNMT consists of three-tier items. Students were asked to choose the correct choice in the first tier, and then to justify their choice of the answer provided in the second tier. Finally, they were asked to indicate the confidence level of their responses in the third tier. Results showed that the PSTs were not competent in their understanding of the PNM concepts. Moreover, some had strongly held misconceptions. Finally, the PSTs struggled to provide coherent answers to the items on the PNMT across three tiers.