Introduction: Serum Cortisol is a glucocorticoid hormone released from the zona fasciculata layer of the adrenal cortex, which plays an important role in regulation of blood pressure and metabolism. Hypothyroidism causes elevated cortisol levels due to both decreased clearance and negative feedback of cortisol on the hypothalamic pituitary-adrenal axis. Aim: To investigate the correlation of serum cortisol and with Triiodothyronine (T3), Thyroxine (T4), Thyroid Stimulating Hormone (TSH) and serum cortisol levels in patients with hypothyroidism. Materials and Methods: Present case-control study was carried out in the Department of Biochemistry, Gandhi Medical College and Hospital, Secunderabad, Telanagana, India, July 2022 to August 2022. A total of 60 patients with hypothyroidism (cases) aged between 35-60 years, and 60 controls were selected from Gandhi Medical College and Hospital. Samples were analysed in Siemens Advia centaur XPT. T3, T4, TSH and cortisol levels were estimated by “CLIA” method. Data were statistically analysed using Karl Pearson’s correlation test. Results: In present study, there were five males and 55 females in control group, 23 males and 37 females were in case group. The mean age in cases was 39.39±6.44 years and in control group was 39.38±10.97 years. The mean T3 in subjects cases was 10.74±5.34 ng/mL, as compared to 8.93±2.78 ng/mL, in controls. The mean TSH level in cases was 13.38±20.43 mIU/L, as compared to 3.47±4.20 mIU/L, in controls. The mean cortisol level in cases was 64.88.44±16.25 µg/dL, as compared to 9.55±5.59 µg/dL, in controls. There was significant positive correlation between serum cortisol level and T3 (r=0.12, p-value 0.02), T4 (r=0.12, p-value 0.02), and TSH Levels ( r= 0.31, p-value 0.01) in case group. Conclusion: The results of present study showed that serum levels of cortisol were significantly increased in hypothyroidism patients as compared to normal healthy controls.