Vitamin D substantially influences sports performance and post-exercise recovery because it offers anti-inflammatory, antioxidant and cellular protective properties. However, deficient levels of 25-hydroxyvitamin D 25(OH)D (25(OH)D) (<30 ng/mL) could impact the health of individuals, lead to musculoskeletal disorders and decrease athletic performance. Therefore, it would be appropriate to know the interactions between genes and vitamin D. We evaluated whether 25(OH)D had a possible connection to the presence of certain SNPs in CYP2R1 (rs10741657), GC (rs2282679) and muscle VDR (rs2228570) genes, with serum 25(OH)D concentrations and the degree of WOD performance in highly trained CrossFit® practitioners. Knowing these relationships could be instrumental for personalized vitamin D supplementation and training strategies. Using a standardized commercial enzyme-linked immunosorbent assay procedure, the concentrations of 25(OH)D were determined and the genotyping procedures for each SNPs were carried out using specific assays with the KASpar® test. The 25(OH)DA performance level in grades was established based on the CrossFit® Total score (sum in kilograms of one Repetition Max Squat, Press and Deadlift). Significant differences (p < 0.05) in 25(OH)D concentration were found between each of the SNPs of CYP2R1 and GC with 25(OH)D. We discovered statistically significant weak positive correlations (p < 0.05) between 25(OH)D and AA-alleles of the CYP2R1 and VDR genes, and TT-alleles of the GC gene. Additionally, AA (rs10741657 and rs2228570) and TT (rs2282679) have a probability between 2 and 4 of having major concentrations of 25(OH)D and 25(OH)D25(OH)D. Conversely, GG alleles present a probability of suboptimum values of 25(OH)D of 69%, 34% and 24% for VDR, GC and CYP2R1, respectively, showing a strong moderate positive correlation (r = 0.41) between the degrees of sports performance and 25(OH)D25(OH)D plasma levels. The different polymorphisms of our three candidate genes CYP2R1 (rs10741657), GC (rs2282679) and VDR (rs2228570) disturb 25(OH)D concentration and play a critical role in the sports performance of elite CrossFit® practitioners. These results could highlight that the evaluation of genetic factors is key to designing a vitamin D supplementation strategy to improve sports performance. [ABSTRACT FROM AUTHOR]