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Background Although no gold standard exists, liquid chromatography tandem mass spectrometry (LC–MS/MS) is a precise and accurate method for the analysis of plasma 25-hydroxyvitamin D (25(OH)D). Immunoassays are more readily available and require small volume sampling, ideal for infant testing. The objective was to compare two commercially available immunoassays for measuring circulating 25(OH)D concentration in infant plasma against LC–MS/MS. Methods Capillary blood samples from 103 infants were analyzed for plasma 25(OH)D using an enzyme immunoassay (EIA, Octeia, IDS Ltd.) and radioimmunoassay (RIA, DiaSorin). Plasma 25(OH)D 3 , C-3 epimer of 25(OH)D 3 (3-epi-25(OH)D 3 ) and 24,25-dihydroxyvitamin D (24,25(OH) 2 D 3 ) were measured on the same samples using LC–MS/MS. To establish whether plasma 24,25(OH) 2 D 3 or 3-epi-25(OH)D 3 interferes with these immunoassay results, the zero 25(OH)D calibrator from each assay kit was spiked with increasing amounts of 24,25(OH) 2 D 3 or 3-epi-25(OH)D 3 . Results Classifying infants below the common vitamin D status targets of 50 nmol/L and 75 nmol/L respectively, 58% and 99% fell below using the RIA, 19% and 56% with the EIA and 31% and 76% with LC–MS/MS. Compared to LC–MS/MS, both immunoassays showed poor Bland–Altman limits of agreement for 25(OH)D concentrations (RIA: limits of agreement − 27 to + 13%; EIA: − 12 to + 41%), and mountain plots (folded cumulative distribution) depicted significant skew and bias. Spiked 24,25(OH) 2 D 3 concentrations, but not 3-epi-25(OH)D 3 , appeared as > 100% of known values on the EIA but not on the RIA thus, suggesting that the EIA may cross-react with 24,25(OH) 2 D 3 to a greater extent than 3-epi-25(OH)D 3 . Conclusion Two common immunoassays resulted in very different classifications of vitamin D status possibly related to the interference of other vitamin D metabolites. Based on these data, LC–MS/MS assessment of vitamin D status is recommended in young infants (4–6 weeks of age).