Vitamin D can favor the state of iron metabolism, and testosterone and cortisol as an indicator of the athlete's muscle recovery and therefore improve sports performance. Deficient levels of 25-hydroxyvitamin D (25(OH)D) (<30 ng/mL) may compromise health, lead to musculoskeletal disorders, and decrease athletic performance. We evaluated the impact and presence of a possible linkage between Single Nucleotide Polymorphisms (SNPs) CYP2R1 (rs10741657), GC (rs2282679), and VDR (rs2228570), serum 25-OH/D concentrations and the link with the degree sports performance in 19 elite cyclists’ men not supplemented with vitamin D Levels of 25-OH vitamin D were quantified with a commercial enzyme-linked immunosorbent assay kit and 3 SNPs were genotyped with KASPar assays. A performance level was established based on the sum of three physical performance tests such as test time, average power, and blood lactate level. Significant differences in 25-OH/D concentration were determined between the bi-allelic combinations of rs228679 and rs228570. We detected statistically significant weak positive correlations between the AA (rs10741657 and rs228570) and TT (rs228679) and alleles and 25-OH/D and the probability of having higher 25-OH/D concentrations was 2- to 3-fold higher. However, the GG alleles of the 3 SNPs showed that the probability of having optimal 25-0H/D concentrations decreases by 32% for rs10741657, 38% for rs228679, and 74% for rs228570, showing a strong negative correlation between the level of sports performance and 25-OH/D levels. Allelic variations in CYP2R1 (rs10741657), GC (rs2282679), and VDR (rs10741657) affect vitamin D levels and decisively influence sports performance in elite athletes. These results could highlight that the evaluation of genetic factors is key to designing a vitamin D supplementation strategy to improve sports performance.