P-Vitamin B12 is a commonly used biochemical test. Evaluation of test results and diagnosis of vitamin B12 deficiency are challenging, and the role of different biochemical methods remains unclear.The aim of this study was to establish reference intervals for plasma vitamin B12 concentration using different immunoassays (method 1: Alinity, Abbott Laboratories; method 2: Cobas 6000, Roche Diagnostics; method 3: Atellica IM, Siemens Healthineers). Direct reference intervals were established among blood donors (n = 129) and indirect reference intervals among adult patient results of plasma vitamin B12 concentration requested by general practitioners in the North Denmark Region from 15 August to 15 October 2022 (n = 34,181). Finally, the frequency of low vitamin B12 concentration using different uniform cut-offs was evaluated.Direct reference intervals (2.5-97.5 percentiles) were as follows for method 1: 168-553 pmol/l; method 2: 202-641 pmol/l; and method 3: 211-551 pmol/l. Indirect reference intervals were as follows for method 1: 133-541 pmol/l; method 2: 172-619 pmol/l; and method 3: 182-162-206 pmol/l. When different cut-offs were applied to patient results, the frequency of having a vitamin B12 concentration below 250 pmol/l differed by biochemical method: 33% (method 1), 17% (method 2) and 14% (method 3).Measurement of plasma vitamin B12 concentration using different immunoassays revealed results and reference intervals that were not interchangeable. Clinical guidelines for the diagnosis of vitamin B12 deficiency should consider the biochemical methods used.None.None.