Preserving the sperm of stallions with significant genetic worth is a widely accepted procedure. Thus, the species' typical reproductive behaviour is altered to facilitate the retrieval of sperm for the use of assisted reproductive biotechnologies, like artificial insemination and cryopreservation of sperm. As the Marwari horse breed is a native breed of our country, the present experiment was designed to define the basic semen parameters of Marwari stallions. The experiment was conducted on six healthy Marwari horses aged between 50 and 140 months for three weeks with a semen collection frequency of twice a week. A total of thirty-six semen samples (six from each horse) were collected using an artificial vagina. Following a macroscopic examination, fresh semen samples underwent a microscopic evaluation. The appearance of fresh semen was milky white or creamy white and the consistency was thin to thick. Fresh seminal pH was 7.54±0.03. Total ejaculated semen volume, gel volume and gel-free semen volume were 67.22±5.77 ml, 14.08±1.98 ml and 53.14±4.16 ml, respectively. Progressive sperm motility of sperm was 78.99±0.65%. Sperm concentration was 195.50±5.67 million/ml. Viability and total morphological abnormalities of sperm were 85.82±0.48% and 4.42±0.23%, respectively. Plasma membrane integrity (HOST), acrosome integrity and DNA integrity of sperm were 53.65±0.85%, 87.36±0.36% and 95.83±0.24%, respectively. A significant difference was observed among Marwari stallions for semen volume (total ejaculated, gel and gel-free) (P<0.05), progressive motility (P<0.05), viability (P<0.05), concentration (P<0.05), plasma membrane integrity (HOST) (P<0.05), acrosome integrity (P<0.05), and DNA integrity (P<0.05) of sperm. Non-significant difference was observed for seminal pH (P>0.05) and total sperm morphological abnormalities (P>0.05).