Although dehydration of ≥ 2% body weight (BW) loss significantly impairs endurance performance, dehydration remains prevalent among athletes and may be owing to a lack of knowledge in relation to fluid requirements. The aim of this study was to assess the hydration status of university/club level athletes (n = 430) from a range of sports/activities (army officer cadet training; bootcamp training; cycling; Gaelic Athletic Association camogie, football and hurling; golf; hockey; netball; rugby; running (sprinting and endurance); Shotokan karate and soccer) immediately before and after training/competition and to assess their nutritional knowledge. Urine specific gravity (USG) was measured immediately before and after exercise and BW loss during exercise was assessed. Nutritional knowledge was assessed using a validated questionnaire. 31.9% of athletes commenced exercise in a dehydrated state (USG >1.020) with 43.6% of participants dehydrated posttraining/competition. Dehydration was particularly prevalent (>40% of cohort) among karateka, female netball players, army officer cadets, and golfers. Golfers that commenced a competitive 18 hole round dehydrated took a significantly higher number of strokes to complete the round in comparison with their euhydrated counterparts (79.5 ± 2.1 vs. 75.7 ± 3.9 strokes, p = .049). Nutritional knowledge was poor among participants (median total score [IQR]; 52.9% [46.0, 59.8]), albeit athletes who were euhydrated at the start of exercise had a higher overall score in comparison with dehydrated athletes (55.2% vs. 50.6%, p = .001). Findings from the current study, therefore, have significant implications for the education of athletes in relation to their individual fluid requirements around exercise.