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While iron deficiency is commonly discussed in populations of professional female athletes, less is known about highly trained, sub-elite female athletes (e.g., those winning international age-group competitions) who generally have less access to medical and allied health support. Thirteen non-professional highly trained female endurance athletes provided training diaries and completed a blood test, where iron markers of haemoglobin (Hb), haematocrit (Hct), C-reactive protein (Crp), serum iron, serum ferritin, and transferrin were assessed. Resting metabolic rate (RMR) and body composition using dual-energy X-ray absorptiometry (DXA) were also obtained. Participants were classified as iron deficient (ID) if serum ferritin was <30 ug/L serum ferritin. Six of the 13 females were classified as ID. Serum iron, ferritin, Hb, Hct, and ferrin were greater in the ID group (p < 0.05). Crp resulted in large to very large correlations with serum iron (r = −0.72), serum ferritin (r = −0.66), and transferrin (r = 0.70). In this population of highly trained female athletes, 46% were diagnosed with sub-optimal iron levels, which could have lasting health effects and impair athletic performance. The need for more education and support in non-professional athletes regarding iron deficiency is strongly advised.