Andrew Watson and Sarah Fowler - the children of the late Dick Watson - have given up their shareholding in the club, with the former also standing down as vice chairman and director.
Any liabilities and loans owed to the family have been written off, while both will continue to have ambassadorial roles within the club.
It brings to an end the family’s 14-year ownership of Rovers, which started when Dick Watson bought into the club alongside Terry Bramall to create the triumvirate with John Ryan which helped to bring a period of great success for Rovers.
“As a family we have been proud to be part of Doncaster Rovers for more than 15 years,” Andrew Watson said in a statement.
Doncaster Rovers defender ruled out for one month
Doncaster Rovers: Former recruitment boss joins Watford in similar role
The difference of opinion and slice of luck that led Jack Degruchy to Doncaster Rovers
Doncaster Rovers player ratings from the dramatic draw at AFC Wimbledon
Why Doncaster Rovers feel the best is yet to come despite a ‘solid start’
“We have loved every minute of our time as directors and I know Dad did too.
“We have some truly wonderful memories and moments such as Cardiff, Wembley and Brentford will be cherished by our family forever.
“However, from a personal perspective, since the passing of Dad the pressure of being an owner and the enjoyment of being a fan have been hard to combine.
“This is something we have discussed openly with Terry, David and Gavin over the past two years and our resignation has been a discussed move during that time.
“We are incredibly proud of what the club has achieved both on and off the pitch during the past two decades, including multiple promotions, cup runs and of course the significant impact the club has made in the community through the Foundation and the Sports College.
“We wish to thank all Doncaster Rovers fans for their continued support and of course Terry, David and Gavin and their families.”
The Tickhill-based Watson family have invested more than £14million in the club, according to Rovers.
Siblings Andrew and Sarah each latterly held a 12.5 per cent stake in Club Doncaster Limited, the parent organisation which owns more than 98 per cent of Doncaster Rovers Limited.
That combined 25 per cent shareholding in the club will now be split between Bramall, CEO Gavin Baldwin and chairman David Blunt following their departures
Following the death of Dick Watson in 2017, his 49 per cent share in Club Doncaster passed to his wife Marion.
A reshuffling of the Club Doncaster shareholding with the Watson and Bramall stakes being reduced and those of Baldwin and increasing, to give all four parties a 25 per cent stake each. Last year, Marion split her share between Andrew and Sarah.