Vitamin D deficiency is frequent in chronic kidney disease (CKD) and hemodialysis (HD) patients. The ‘Kidney Disease Improving Global Outcomes’ (KDIGO) clinical practice guideline for the evaluation and management of chronic kidney disease recommends the administration of vitamin D in these patients for obtaining 25-hydroxyvitamin D (25OHD) serum levels between 30 and 60 ng/mL. Recent studies suggest that 25OHD levels can differ between assays depending on the vitamin D status of the patients, the reference method used being liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). As determination of 25OHD levels is crucial for CKD and HD patients, our aim was to establish whether the results obtained, using immunoassay and mass spectrometry, were similar for these populations. 25OHD serum levels were measured in 136 subjects (46 regardless of their disease, 30 with normal kidney function, 30 with CKD and 30 with HD) using two immunoassays, two LC-MS/MS methods, and one HPLC-UV method. The 25OHD levels obtained using LC-MS/MS and immunoassay methods did not differ significantly in the overall patient cohort, but higher values were observed using LC-MS/MS in CKD and HD patients, thereby leading to a different clinical interpretation. Discrepancies between LC-MS/MS and immunoassays were not explained on the basis of interference with C₃-epi-25OHD or alfacalcidol treatments. It is crucial to standardize the commercial assays used for 25OHD measurement and/or to determine the best target values for CKD and HD patients, depending on the method used.