This study was to use differential item functioning (DIF) analysis to examine if there were items in the Mental, Emotional, and Bodily Toughness Inventory (MeBTough) 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 MeBTough items. Using Mantel-Haenszel and SIBTEST methods, 43 items were analyzed for DIF. Four MeBTough items were identified as large DIF items by both Mantel-Haenszel and SIBTEST methods, where item 21 favored non-athletes, item 40 favored athletes, item 2 favored males, and item 17 favored females. Athletic membership DIF disappeared whereas gender DIF still existed at the scale level. Overall, there are gender and athletic membership DIF items in the MeBTough, but only gender DIF still exists at the scale level. Thus, conclusions regarding gender differences in mental toughness should be made with caution when using total MeBTough scores.