It is believed the three dead are Buddhist monks. It is not yet known if they had links to the Samye Ling Tibetan Centre near Dumfries.
Emergency services were called to the A68 Pathhead to Soutra road at around 7.40am this morning.
One of the vehicles was upside down with the roof crushed.
One person has been taken to Edinburgh Royal Infirmary and is being treated for serious injuries.
The road was closed southbound and Lothian and Borders Police were diverting traffic via the B6370 Gorebridge road.
A Nissan Note travelling northbound was involved in the collision with a Skoda Octavia being driven in the opposite direction.
The three back seat passengers of the Nissan, who were all male, died at the scene.
The driver and front seat passenger, a woman and a man respectively, have been taken to Edinburgh Royal Infirmary for treatment to their injuries, which are not believed to be life-threatening.
The male driver of the Skoda has also been taken to ERI, although he has not suffered any serious injury.
Inspector Simon Bradshaw, from the roads policing unit at Lothian and Borders Police, said: "This is a tragic incident, and we are currently in the process of carrying out inquiries in order to establish the full circumstances of the collision.
"I am appealing to anyone who noticed the gold Nissan Note or the silver Skoda Octavia on the A68 this morning to get in touch."
Yesterday two people were killed in a head-on crash not far from today's accident.
A 53-year-old man and a 51-year-old woman were travelling south in a silver Kia when it collided with a red Hyundai I30 driving north.
The woman died at the scene of the accident and the man died in Edinburgh Royal Infirmary where he had been taken for treatment, Lothian and Borders Police said.
Two people travelling in the Hyundai were also taken to hospital after the incident on the A697 at Carfraemill, in the Scottish Borders, at around 2.55pm yesterday.