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Multiple choice items (MCIs) are commonly used in high-stake testing and classroom assessment because of their reliable assessment results. However, the recent literature has revealed that item-writing guidelines have been repeatedly violated especially for the MCIs, and it can threaten test validity. Another threat to the validity occurs when items favor certain groups (e.g., female students) even though the underlying ability of the different groups are the same, and it is called differential item functioning (DIF). This empirical study aims to examine whether the violation of item-writing guidelines for MCIs can cause DIF for gender differences. The findings showed that two MCIs with negative stem and one complex MCI showed DIF effect based on the final test for a course.