Background: Vitamin D deficiency during pregnancy has been associated with increased risk of complications and adverse perinatal outcomes. We evaluated seasonal variation of 25-hydroxyvitamin D [25(OH)D] among pregnant women, focusing on patterns and determinants of variation. Methods: Data came from three cohort studies in the US that included 2583 non-Hispanic Black and White women having prenatal 25(OH)D concentrations determined. Fourier time series and generalised linear models were used to estimate the magnitude of 25(OH)D seasonality. We modelled seasonal variability using a stationary cosinor model to estimate the phase shift, peak–trough difference, and annual mean of 25(OH)D. Results: We observed a peak for 25(OH)D in summer, a nadir in winter, and a phase of 8 months, which resulted from fluctuations in 25(OH)D3 rather than 25(OH)D2. After adjustment for covariates, the annual mean concentrations and estimated peak–trough difference of 25(OH)D among Black women were 19.8 ng/mL [95% confidence interval (CI) 18.9, 20.5] and 5.8 ng/mL [95% CI 4.7, 6.7], and for non-Hispanic White women were 33.0 ng/mL [95% CI 32.6, 33.4] and 7.4 ng/mL [95% CI 6.0, 8.9]. Conclusions: Non-Hispanic Black women had lower average 25(OH)D concentrations throughout the year and smaller seasonal variation levels than non-Hispanic White women. This study’s confirmation of 25(OH)D seasonality over a calendar year has the potential to enhance public health interventions targeted to improve maternal and perinatal outcomes.