Measurement of vitamin D status has significant use in clinical and research settings, including during pregnancy. We aimed to assess the agreement of total 25-hydroxyvitamin D (25(OH)D) concentration, and its three analytes (25-hydroxyvitamin D3 (25(OH)D3 ), 25-hydroxyvitamin D2 (25(OH)D2 ) and Epi-25-hydroxyvitamin D3 (Epi-25(OH)D3 )), in plasma and serum samples collected during pregnancy, and to examine the proportion of women who change vitamin D status category based on sample type. Matching samples were collected from n = 114 non-fasting women between 12–25 weeks gestation in a clinical trial in Newcastle, Australia. Samples were analysed by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) to quantify total 25(OH)D and its analytes and examined using Bland-Altman plots, Pearson correlation (r), intraclass correlation coefficient and Cohen’s Kappa test. Serum total 25(OH)D ranged from 33.8–169.8 nmol/L and plasma ranged from 28.6–211.2 nmol/L. There was a significant difference for total 25(OH)D based on sample type (measurement bias 7.63 nmol/L for serum vs plasma (95% Confidence Interval (CI) 5.36, 9.90, p ≤ 0.001). The mean difference between serum and plasma concentrations was statistically significant for 25(OH)D3 (7.38 nmol/L; 95% CI 5.28, 9.48, p ≤ 0.001) and Epi-25(OH)D3 (0.39 nmol/L; 95% CI 0.14, 0.64, p = 0.014). Of 114 participants, 28% were classified as vitamin D deficient (<50 nmol/L) or insufficient (<75 nmol/L) based on plasma sample and 36% based on serum sample. Nineteen (16.7%) participants changed vitamin D status category based on sample type. 25-hydroxyvitamin D quantification using LC-MS/MS methodology differed significantly between serum and plasma, yielding a higher value in plasma; this influenced vitamin D status based on accepted cut-points, which may have implications in clinical and research settings.