The study was conducted to investigate the impact of different housing systems on the development of equine behavior through variations in serum cortisol concentrations. N=120 x TBP (Thoroughbred Pakistan) of three different groups (A=40 horses 2years not gelded, B= 40 age 3 years and C= 40 four years gelded) and four distinct types of housing systems. Single Housing System No Interaction (SHSNI), the horses didn’t have any interaction among them, Single Housing System Limited Interaction (SHSLI), Double Housing System (DHS), a pair of horses were kept in house and Group Housing System (GHS), where (n=40) horses were having complete interaction among them, were considered in the study. The study was conducted on Remount Depot Mona (known as Home of Horses) which is in Mandi Bahuddin, Punjab, Pakistan. The duration of study was 120 days and conducted in four phases (Phase-I SHSNI, Phase-II SHSNI, Phase- III DHS and Phase- IV GHS were used) and each phase was comprised of 30 days duration. Before shifting to any housing system, the blood was collected to measure cortisol concentration at zero day and then after every 30^th day again blood was collected prior to shifting to next housing system. Blood was collected from each horse for serum cortisol analysis through Cortisol kit. Competitive Enzyme immunoassay (CEIA) was used for quantification of cortisol in samples by using commercially available cortisol ELISA kits. Analysis of variance by considering randomized complete block design was performed for statistical analysis. The cortisol concentration exhibited no significant fluctuations within the groups, but a noteworthy disparity was observed between the housing systems (P<0.05). The cortisol concentration measured at 0^th day was 1.53 µg/dL which was decreased 0.58 µg/dL in SHSNI and increased 0.59 µg/dL in SHSLI reached slightly above the average value at 0^th day, then it decreased (0.57 µg /dL) in DHS and again increased in GHS (0.58 µg /dL) and reached to 1.55 µg /dL. The study concludes that SHSLI is a more suitable housing system than all others, needed to be adopted for the better welfare of the horses than other housing systems. This house has maximum social interaction, less stress and stereotypes with limited injuries as compared to alternative housing systems. Further investigation can be performed to standardize that type of housing which maintains the minimum stress level of Thoroughbred horses.