David Denton, Lee Jones and Jim Hamilton will all miss Scotland's summer tour to Australia, Fiji and Samoa.
It was revealed on Wednesday that Denton and Jones had both been ruled out due to injury. Jones has a fractured right shoulder blade, while his Edinburgh team-mate Denton has damage to his left ankle ligaments.
The news came shortly after Gloucester lock Hamilton had been handed a seven-week ban following a brawl with London Irish hooker David Paice during the Aviva Premiership game between the clubs last Saturday.
Denton and Hamilton were ever-presents during the Six Nations, while Jones only missed the final match in Italy due to concussion, and their absences are blows for a Scotland side seeking to arrest their worst run since 1998.
Scotland go into the Test with Australia on June 5 seeking to end a seven-match winless sequence, before playing the NSW Waratahs on June 10 and Tests against Fiji on June 16 and Samoa a week later.
Head coach Andy Robinson is poised to name his squad on May 16.
Jones' absence can be compensated for by the inclusion of Edinburgh's flying Dutch winger Tim Visser, who come June 12 will be eligible for Scotland on the grounds of three years' residency.
Missing Denton will stretch Scotland's back-row resources, with Saracens' Kelly Brown already absent after he dislocated his fibula in January, while Hamilton's indiscretion means he returns behind World Cup captain Alastair Kellock in the second-row order.
Jones and Denton, who have four and six caps respectively, proved their Test credentials in their maiden Six Nations as Scotland finished with the Wooden Spoon, while also performing strongly for Edinburgh as the capital club reached the Heineken Cup semi-final.
Scotland team doctor James Robson said: "Lee suffered the fracture in the Heineken Cup semi-final loss to Ulster last month.
"Injuries of this nature typically resolve over the course of six to eight weeks, so he will not be available for the tour.
"David sustained the ankle injury in the final game of the league season against Treviso last weekend and subsequent scanning of the joint revealed damage to the ligaments.
"Following consultation with John McKinley, the ankle specialist at Spire Murrayfield Hospital, David will not require surgery but will now embark on a prolonged period of rehabilitation which will rule him out of the summer tour.
"His estimated return would be between eight to 10 weeks."
Both Hamilton and Paice were handed seven-week suspensions by a three-man Rugby Football Union panel.
Both were sent off by referee Dave Pearson following an incident that sparked a mass brawl.
Both players were shown yellow cards following an initial skirmish, and they were walking off the pitch together when Hamilton punched his opponent.
Paice, who required stitches in a head wound, reacted and Pearson had no option but to brandish red cards.
Hamilton's ban will run until August 19, sidelining him for the Scotland tour and Gloucester's warm-up games for next season.
Paice, meanwhile is suspended from August 10 to September 23, meaning he will miss pre-season fixtures and up to four weekends of Premiership action.
Both players can appeal.