The purpose of this study was to investigate how mental rotation strategies affect the identification of chemical structural formulas. This study conducted event-related potentials (ERPs) experiments. In addition to the data collected in the ERPs, a Chemical Structure Conceptual Questionnaire and interviews were also administered for data collection. Eighteen university students majoring in chemistry were recruited. In the ERP experiments, the participants were required to identify 2D figures, 2D chemical structural formulas, 3D objects and 3D chemical structural formulas. The contours of 2D figures are similar to those of 2D chemical structural formulas, but they contain no content knowledge. Likewise, the contours of 3D objects are similar to 3D chemical structural formulas without content knowledge. The results showed that all students used similar strategies of mental rotation in identifying 2D figures, 3D objects and 3D chemical structural formulas. However, the high-achieving students used different strategies in identifying 2D figures and chemical structural formulas, while the low-achieving students tended to use similar strategies of mental rotation in identifying both 2D figures and chemical structural formulas. The results indicate that some of the difficulties in identifying 2D chemical structural formulas that students encounter are due to their inappropriate strategies of mental rotation.