The US Institute of Medicine defined serum 25-hydroxyvitamin D (25OHD) cut point values of 30 nmol/L and 40 nmol/L were used to assess the vitamin D status of South Asian and European Canadians of self-identified ancestry living in the National Capital Region of Canada. Serum 25OHD values were measured in the spring and fall of 2012 to represent status during the winter and summer months, respectively. A total of 1238 measurements were obtained from 669 participants (49% South Asian ancestry): some participants were measured only once (spring or fall). Median 25OHD values were significantly higher in participants of European ancestry: 70.8 nmol/L (68.1, 73.5; 95% CI) versus South Asian ancestry: 42.7 nmol/L (40.5, 45.0; P<0.001). Spring vs. fall differences were small for each ethnic group and significant only for those of European ancestry (2.9, CI: 1.0-4.9 nmol/L; P = 0.01). Among participants of South Asian ancestry, 27.3% (fall) and 29.1% (spring) of females had values <40 nmol/L while the percentages for males were considerably higher (36.5% and 44.2%, respectively). The corresponding values for participants of European ancestry were ≤10%, showing that the South Asian participants were less likely to achieve the 25OHD concentrations established by the IOM for optimum bone health. Investigation of the factors related to serum 25OHD levels showed that supplement intake and ethnic background were associated with the biggest differences. Skin color was not a major factor, suggesting that genetic factors are responsible for the observed differences between participants of different ethnic backgrounds.