In this study the authors used differential item functioning analysis to examine if there were items in the Mental, Emotional, and Bodily Toughness Inventory functioning differently across gender and athletic membership. A total of 444 male (56.3%) and female (43.7%) participants (30.9% athletes and 69.1% non-athletes) responded to the Mental, Emotional, and Bodily Toughness Inventory items. Using Mantel-Haenszel and simultaneous item bias test methods, 43 items were analyzed for differential item functioning. Four Mental, Emotional, and Bodily Toughness Inventory items were identified as large differential item functioning items by both Mantel-Haenszel and simultaneous item bias test methods, where item 21 favored non-athletes, item 40 favored athletes, item 2 favored males, and item 17 favored females. Athletic membership differential item functioning disappeared, whereas gender differential item functioning still existed at the scale level. Overall, there are gender and athletic membership differential item functioning items in the Mental, Emotional, and Bodily Toughness Inventory, but only gender differential item functioning still exists at the scale level. Thus, conclusions regarding gender differences in mental toughness should be made with caution when using total Mental, Emotional, and Bodily Toughness Inventory scores. (Contains 4 tables and 2 figures.)