Evidence suggests that low concentrations of 25-hydroxyvitamin D(3) (25OHD3) during gestation may be associated with a range of adverse health outcomes in later life. Retrospective estimation of perinatal vitamin D status using questionnaires is extremely unreliable and stored serum samples are rarely available. We aimed to validate the use of dried blood spots (DBS) to estimate perinatal vitamin D status and to determine whether inter-group differences in cord serum 25OHD3 are reflected in DBS. We examined 25OHD3 in 4-year-old frozen cord sera and matched DBS from neonates born at a hospital in Melbourne, Australia (n = 100). We examined the correlation between these values and also investigated whether the expected seasonal (winter/spring vs. summer/autumn) difference in serum 25OHD3 was reflected in DBS values. 25OHD3 was assayed in triplicate using tandem mass spectroscopy in both a 3 microL sample of cord serum and in matched 3 mm punches from archived DBS. 25OHD3 concentrations in neonatal cord serum and DBS were highly correlated (r = 0.85, P < 0.0001). As expected, serum 25OHD3 concentrations were higher in neonates born in summer/autumn (December to March) vs. winter/spring (April to November) (median 46.6 vs. 23.7 nmol/L, P < 0.0001). A comparable difference was seen in DBS values (17.8 vs. 10.5 nmol/L, P = 0.0001). Archived DBS samples provided a valid measure of perinatal vitamin D status and identified inter-seasonal differences in perinatal 25OHD3 concentrations. They could be used for case-control studies investigating the association between perinatal vitamin D status and later health outcomes.