Athletic trainers commonly work with athletes of the opposite gender yet it is not fully understood if gender may influence these interactions. The purpose of this study was to determine the extent that gender influences comfort, communication, and trust in the athlete and athletic trainer relationship. A 26-item survey containing Likert based questions and open-ended and a trust instrument were distributed in athletic training rooms to analyze comfort, communication, and trust. Comfort, communication, and trust were significantly lower when working with athletic trainers of the opposite gender. Athletes reported trust and communication as the most valued aspect of the relationship with their athletic trainer. Despite lower perceived scores, 150 out of 178 participants reported no preference for the gender of their athletic trainer. Athletic trainers should understand that athletes may experience a decrease in comfort, communication, and trust when working with athletic trainers of the opposite gender. Key Words: Comfort, Trust, Communication, Athletic Trainer