Athletes may experience similar comfort concerns as patients in other allied health care fields. The purpose of this study was to address athlete comfort with injury and condition care provided by same- and opposite-sex athletic trainers. In this cross-sectional survey design, three National Collegiate Athletic Association Division I university athletic programs were invited and agreed to participate. Universities were selected by geographic location and affiliation with three distinct National Athletic Trainers' Association districts. A total of 685 athletes (341 women, 344 men) completed questionnaires (277 in the Mid America District, 282 in the Far West District, and 126 in the Southeast District). The Gender Comfort with Athletic Trainer Questionnaire consisted of 17 injuries and conditions common to both female and male athletic trainer scenarios. Three gender-specific items were added to each scenario. Responses were scored on a 5-point scale anchored by 1 (very uncomfortable) and 5 (very comfortable). Participants were also asked to indicate the reason for any degree of discomfort. Internal consistency, determined by Cronbach alpha, was .93 for the female athletic trainer scenario and .95 for the male athletic trainer scenario. Results indicated significant injury and condition category by sex interactions for general medical conditions (F1,683 = 578.9, P < .001), psychological conditions (F1,683 = 136.2, P < .001), injuries to the upper body (F1,683 = 175.7, P < .001), injuries to the mid-body (F1,683 = 199.1, P < .001), and injuries to the lower body (F1,683 = 4.9, P < .001). For gender-specific injuries and conditions, a difference was found between the mean ratings in both female athlete comfort (t340 = -26.350, P < .001) and male athlete comfort (t340 = -26.350, P < .001) when athletes were provided care by a female athletic trainer and a male athletic trainer. Overall, athletes appeared to be more comfortable when provided care by a same-sex athletic trainer. The most common reason reported for discomfort was gender related. It was concluded that both athletes and athletic trainers should be made aware that athletes do experience discomfort, especially if the injury or condition is intimate in nature. It may be necessary to have athletic trainers of both sexes accessible to athletes to optimize their contribution to the treatment received. Keyword(s): athletic training, gender issues, research