Vitamin D deficiency has become a major public health problem as it is prevalent in all age groups and ethnicities. The higher rates of hypovitaminosis D in the sunniest areas of the world, including the Middle East, Asian, and North Africa countries, such as Libya. Rickets and osteomalacia still occur in this sunny region. Hypovitaminosis D prevails, with rates varying 30–90%. Objectives: The aim of our study was to evaluate the vitamin D status among populations in Alejelat region in Western Libya. Methods: An observational study was conducted among 733 subjects (141 males & 236 Females) in Alejelat region in Western Libya, over a period of six months from first March 2019 to 30th August 2019. The subjects participating in the study were requested to complete a questionnaire that covered socio-demographic data. Serum 25-hydroxyvitamin D (25(OH) D) level was measured using an enzyme immunoassay method. The associations of the levels of 25(OH)D3 with gender, and age groups were assessed throughout using the Chi-square test. Results: Out of 377 subjects; 236 subjects (62.6%) were females, and 141 subjects (37.4%) were males. The higher percentage of subjects were 56.78% in females and 31.21% in males in those aged (21-40) years. The prevalence of vitamin D deficiency was 57.45% in males and 69.49% in females. Overall, the estimated prevalence of severe vitamin D deficiency (˂ 10 ng/ml) was 39.26%, Moderate vitamin D deficiency (10-20 ng/ml) was 25.73%, insufficiency (21-29 ng/ml) was 17.77% and the proportion of the sample population with adequacy of vitamin D concentrations (≥30 ng/ml) was 17.24%. The higher prevalence of vitamin D deficiency were 37.59% moderate vitamin D deficiency in male subjects and 39.26% severe vitamin D deficiency in female subjects.