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Background: Female pattern hair loss is also observed in women without increased androgen levels. Vitamin D is a factor that has recently been considered in dealing with these patients. Deficiency of Vitamin D might be correlated with diffuse hair loss. Objective: To determine the frequency of serum levels of Vitamin D3 in patients with female pattern of hair loss (FPHL) at tertiary care hospital Karachi. Methodology: This cross-sectional research was conducted upon a sample of 163 female patients (chosen via non-probability – consecutive sampling) presenting to the Dept. of Dermatology, Abbasi Shaheed Hospital, Karachi from 29th July 2018 to 28 th January 2019, with a clinical diagnosis of FPHL. Data was recorded onto a structured questionnaire containing inquiries pertaining to basic biodata, sociodemographic details, and inferences obtained from blood tests (CBC, serum ferritin, TSH level and vitamin D level). Serum 25 OH Vitamin D level < 20 was taken as deficient, 21-29 was insufficient and over 30 was sufficient. Data was analyzed using SPSS. v. 21.0. Results: The mean age was 30.39±6.36 years. Mean FPHL duration was 5.85±2.89 months. Total 63.2% patients were classified as mild, 26.45% patients were classified as moderate and 10.4% patients were classified as severe. Total 73% patients were found as vitamin D deficient, 22.1% as vitamin D insufficient and 4.9% as vitamin D sufficient. Significant association of vitamin D deficiency was observed with education status, vitamin D intake since past 6 months, and socio economic status. Conclusion: Results showed high vitamin D3 deficiency (73.0%) among patients with female pattern of hair loss (FPHL).