Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating multi-system disorder whose pathogenesis is associated with metabolic and immune dysfunction. Dysfunction of cholesterol metabolism and vitamin D deficiency are considered potential path-ogenic factors. The DHCR7 gene, encoding 7-dehydrocholesterol reductase, catalyzes the final step in cholesterol biosynthesis and simultaneously determines the availability of provitamin D. Pathogenic DHCR7 mutations, leading to the development of Smith-Lemli-Opitz syndrome (SLOS) in the homozygous state, are characterized by a high prevalence in the population as heterozygous carriers (~1%). Therefore, it was hypothesized that heterozygous carriage of DHCR7 mutations may be associated with an increased predisposition to the development of ME/CFS. Materials and Methods: We used open-source genetic data from the DecodeME project (n = 15,579 ME/CFS patients, 259,909 controls). We analyzed the frequency of 11 pathogenic DHCR7 mutations: IVS8-1G>C, W151X, T93M, V326L, R404C, R352W, E448K, R352Q, G410S, R242C, and F302L. The association with the ME/CFS phenotype was assessed using a χ² test in six datasets (the overall sample (gwas_1), subsamples of men (gwas_1_male), women (gwas_1_female), spontaneous CFS development (gwas_1_non-infectious_onset), CFS development with infectious onset (gwas_1_infectious_onset), and a subsample with a 1:10 patient: control ratio (gwas_2)). Results. Only the IVS8-1G>C mutation was detected in the DecodeME data (carrier frequency ~1%). A statistically significant association (p-value ≈ 0.013) was observed in only one subsample (gwas_2) but was not replicated in the others. The remaining mutations were not detected in the DecodeME data. Conclusions. The obtained results do not support the hypothesis of a link be-tween carriage of SLOS-inducing DHCR7 mutations and ME/CFS. This negative result is im-portant for the correct refinement of metabolic hypotheses regarding pathogenesis and the pri-oritization of research areas. Further study of sterol metabolism and metabolomic biomarkers in patient subgroups is recommended.