Vitamin D deficiency is widespread in different populations and regions worldwide and has become a global health issue. The vitamin D status of the population in the Yunnan Province of Southwest China has not been evaluated to date. Therefore, in this study, we evaluated the vitamin D status according to the serum concentrations of 25-hydroxyvitamin D (25(OH)D) in individuals of Yunnan Province, a low-latitude, high-altitude and multiracial region in China. The data on 25(OH)D concentrations from October 2012 to December 2017 were retrospectively collected and assessed using the laboratory information system from 52 950 hospital-based participants (age, 1 day-96 years; females, 73.74%). The serum concentration of 25(OH)D was evaluated using a chemiluminescent immunoassay. The analysis was stratified by sex, age, sampling season, testing year, minority, residential district, latitude, altitude and meteorological factors. Vitamin D status was classified as follows: severe deficiency: <10 ng/mL; deficiency: <20 ng/mL; insufficiency: <30 ng/mL; and sufficiency: >= 30 ng/mL. The results showed that vitamin D deficiency is highly prevalent in Yunnan Province in a hospital-based cohort, with a deficiency and severe deficiency rate of 65.1% and a sufficiency rate of 5.30%. Significantly lower vitamin D levels and sufficiency rates were observed in females than in males (20.13 +/- 7.22 ng/mL vs. 17.56 +/- 6.66 ng/mL and 8.20% vs. 4.20%; p < 0.01, respectively); in spring and winter (16.93 +/- 6.24 ng/mL; 2.97% and 16.38 +/- 6.43 ng/mL; 3.06%, respectively) than in summer and autumn (20.23 +/- 7.14 ng/mL; 8.02% and 19.10 +/- 6.97 ng/mL; 6.61% [p < 0.01], respectively); and in older individuals (0-6 years: 28.29 +/- 13.13 ng/mL vs. >60 years: 14.88 +/- 8.39 ng/mL; p < 0.01). Relatively higher vitamin D levels were observed in individuals of Yi, Zhuang, Hani, Dai, Miao and Lisu minorities and lower levels in individuals of Hui and Zang minorities compared with those of the Han nationality (p < 0.01). The mean sunlight duration, mean air temperature, maximum ultraviolet value and latitude were significantly correlated with vitamin D levels (r = -0.53, 0.60, 0.31, -0.68, respectively; p < 0.05). These results suggest that vitamin D status is influenced by sex, age, minority, latitude and some meteorological factors in areas with high and low altitudes. Hence, new public health policies, such as advice on sunshine exposure, food fortification and nutrition education, as well as the implementation of vitamin D supplementation programmes must be considered to alleviate vitamin D deficiency in Yunnan province, Southwest China.