Vitamin D is known to be critical for the growth and development of young calves and contributes to activation of innate immune defenses of cattle. The 25-hydroxyvitamin D (25D) metabolite is the precursor to the active vitamin D hormone and its concentration in serum serves as an indicator of an animal’s vitamin D status. Normal serum 25D concentrations originally described for cattle are between 20 and 50 ng/mL, but recent evidence has indicated that serum 25D concentrations below 30 ng/mL are insufficient for adequate health. Because little information is available regarding vitamin D status of beef calves, the objective of this study was to evaluate serum 25D concentrations of beef calves born during the spring calving season. Four separate cohorts of calves were studied where serum samples were collected at birth and again 3–4 weeks after birth or in mid to late summer. Serum 25D concentrations were determined by using a 25D ELISA. In the first cohort, the average (±SD) concentrations of 16 Angus and Brangus calves born in Florida were 25.6 ± 12.1 ng/mL at birth in February and 26 ± 10.3, 53.8 ± 12.9, and 59.5 ± 17.2 ng/mL in March, July, and September, respectively. In the second cohort, the average serum 25D concentrations of 12 Angus and Brangus calves born in Florida were 16.4 ± 11.1 ng/mL at birth in April and 29.4 ± 9.7, 43.8 ± 11.8, and 51.0 ± 12.6 ng/mL in June, July, and September, respectively. In the third cohort, the average serum concentrations of 20 Angus calves born in Minnesota were 13.2 ± 6.4 ng/mL at birth in April and 45.9 ± 7.6 ng/mL in July. In the fourth cohort, the average serum 25D concentrations of 15 Charolais calves born from January to April in Idaho were 3.0 ± 2.2 ng/mL at birth and 10.5 ± 7.7 ng/mL at 3 weeks of age. Averages of the serum 25D concentrations for the dams of the calves in cohorts 2, 3, and 4 at calving were 69.5 ± 16.0, 57.7 ± 8.2, and 53 ± 8.2 ng/mL, respectively. In summary, this study indicates that vitamin D insufficiency (serum 25D < 30 ng/mL) is prevalent in newborn beef calves, and that potential opportunities exist to improve health of newborn calves through vitamin D supplementation.