Omega-3 fatty acids (ω-3 FA) are associated with cardiovascular health, brain function, reduction of inflammation, and several other physiological roles of importance to competitive athletes. The ω-3 FA status of National Collegiate Athletic Association (NCAA) Division I athletes has not been well-described. The purpose of this study was to evaluate the ω-3 FA status of NCAA Division I athletes using dietary and biological assessment methodology. Athletes from nine NCAA Division I institutions from throughout the U.S. (n = 1,528, 51% male, 34 sports represented, 19.9 ± 1.4 years of age) completed a food frequency questionnaire (FFQ) to assess ω-3 FA from diet and supplements. Omega-3 Index (O3i) was evaluated in a sub-set of these participants (n = 298, 55% male, 21 sports represented, 20.0 ± 1.3 years of age) using dried blood spot sampling. Only 6% (n = 93) of athletes achieved the Academy of Nutrition & Dietetics' recommendation to consume 500 mg DHA+EPA per day. Use of ω-3 FA supplements was reported by 15% (n = 229) of participants. O3i was 4.33 ± 0.81%, with no participants meeting the O3i benchmark of 8% associated with the lowest risk of cardiovascular disease. Every additional weekly serving of fish or seafood was associated with an absolute O3i increase of 0.27%. Overall, sub-optimal ω-3 FA status was observed among a large, geographically diverse group of male and female NCAA Division I athletes. These findings may inform interventions aimed at improving ω-3 FA status of collegiate athletes. Further research on athlete-specific ω-3 FA requirements is needed.