Sub-optimal calcium, vitamin D and iron intakes are typical in athletes. However, quantification by dietary intake may be erroneous, with biomarkers providing a more accurate assessment. This study aimed to determine the calcium, vitamin D and iron status of 8 junior (i.e., under-18 [U18]; age 15.5 Â± 0.5 years; height 180.4 Â± 6.7 cm; body mass 81.6 Â± 14.3 kg) and 12 senior (i.e., over-18 [O18]; age 19.7 Â± 1.8 years; height 184.9 Â± 6.9 cm; body mass 97.4 Â± 14.4 kg) male rugby union players, and assess their adequacy against reference values. Fasted serum calcium, 25(OH)D and ferritin concentrations were analysed using enzyme-linked immunosorbent assays during the in-season period (March-April). U18 had very likely greater calcium concentrations than O18 (2.40 Â± 0.08 vs. 2.25 Â± 0.19 mmol.l-1). Differences between U18 and O18 were unclear for 25(OH)D (20.21 Â± 11.57 vs. 29.02 Â± 33.69 nmol.l-1) and ferritin (59.33 Â± 34.61 vs. 85.25 Â± 73.53 Âµg.l-1). Compared to reference values, all U18 had adequate serum calcium concentrations, whereas 33% and 67% of O18 were deficient and adequate, respectively. All U18 and 83% of O18 had severely deficient, deficient or inadequate vitamin D concentrations. Adequate (8%) and optimal (8%) concentrations of vitamin D were observed in O18. All U18 and 75% of O18 had adequate ferritin concentrations. Potential toxicity (17%) and deficient (8%) ferritin concentrations were observed in O18. Vitamin D intake should be increased and multiple measures obtained throughout the season. More research is required on the variation of micronutrient status.