Vitamin D deficiency is associated with poor bone health, colorectal cancer, type 1 diabetes and multiple sclerosis. Two national health-related societies in Canada have made recommendations for vitamin D supplementation, yet little research has been reported on the vitamin D status of Canadians. Lifestyle changes, such as sunscreen use, spending less time outdoors and insufficient intake of vitamin D-containing foods as well as northern latitude, may be affecting human vitamin D status. A cross-sectional analysis of 25-hydroxyvitamin D [25-(OH)D] was conducted in pregnant women, newborns (umbilical cord blood) and children. Samples were analysed by liquid chromatography mass spectrometry. Published ranges for 25-(OH)D were used to determine vitamin D status. The prevalence of 25-(OH)D deficiency for the three groups studied revealed most concentrations in the 25-(OH)D deficiency or insufficiency ranges. There were significant differences in all groups studied between seasons, with the exception of maternal blood and female cord blood samples. 25-(OH)D insufficiency was common in all groups for winter and summer, more so in winter. 25-(OH)D insufficiency was common in the three groups studied. The Newfoundland and Labrador population may be at increased risk for vitamin D insufficiency because of factors such as northern latitude and lifestyle issues. Further research on the vitamin D status of this population is important, considering the potential adverse health-related outcomes and the recommendations on supplementation being made.