Background: Many studies show a high prevalence of vitamin D deficiency across various populations the world over. There is relative lack of prevalence data in Southern Punjab, Pakistan. This cross-sectional study is carried out to assess the prevalence of vitamin D deficiency in Southern Punjab population of Pakistan. Study Design: Single centre, Cross sectional study. Place and Duration of the Study: The study was conducted from June 2014 to December 2015, at diagnostic labs of MINAR, Multan, Pakistan. Materials and Methods: 450 subjects were tested for vitamin D. About 3 ml bloods from all the study subjects were collected in EDTA tube and plasma was separated via centrifugation at 4,000 rpm for 5 minutes. The test principle is based on chemiluminescent emission which is measured by photomultiplier. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with Procell. Statistical analysis was done using Chi-square test. Results: Out of a total of 450 subjects tested for vitamin D, females were 386(85.77%) and males were 64(14.22%) of the total sample size. The mean age of respondents was 44.53 years (age range 10-90 years) .Levels of 25(OH)D level in ng/ml was measured in four categories. In total no’s (both males and females) the vitamin D deficient range (<10ng/ml) was 148(32.88%), the insufficient range(10-19.9ng/ml) was 173(38.44%), the inadequate range(20-29.9ng/ml) was 70(15.55%) and desirable range(30-100ng/ml) was 59(13.11%). A high overall prevalence (86.87%) of vitamin D deficiency was observed in the study subjects. In the total subjects the deficiency of vit D observed was 88.33% in females and 78.11% in males. A significant gender-specific difference was also recorded at the cut-off level of 25 (OH) D, with women showing higher prevalence of deficiency compared to men (P < 0.05). Vitamin D level in age wise distribution showed that deficiency was higher in persons in age ranging from 50-90years as compared to age ranging from 10-49 years (P=0.11) Conclusion: The study showed a high percentage of vitamin D deficient individuals. The frequency of vitamin D deficiency was greater in females as compared to males. The vit D deficiency was higher in people above 50y. There is a need to take immediate measures to tackle this growing public health problem.