The Fundamental Concepts for Organic Reaction Mechanisms Inventory (FC-ORMI) is a concept inventory with most items in a two-tier design in which an answer tier is followed by a reasoning tier. Statistical results provided strong evidence for the validity and reliability of the data obtained using the FC-ORMI. In this study, differential item functioning (DIF) was examined to strengthen the validity of the data obtained using the inventory by assessing whether students are scored differently on individual items of the FC-ORMI by gender groups and students' major of study although they are equally proficient in the content. A DIF analysis was conducted using three different methods: Mantel-Haenszel (MH) statistics, logistic regression for uniform and nonuniform DIF, and Rasch analysis. The results indicated that one item showed uniform DIF by gender and one item showed uniform DIF by students' major of study. Additionally, one item showed non-uniform DIF by students' major of study, which was detected by logistic regression, but the graphical evidence did not support it. These results suggest that the FC-ORMI is overall a fair assessment with only one item showing DIF based on gender and one item showing DIF based on students' major of study.