Workers on South Western Railway have voted heavily in favour of striking in the dispute over the role of guards on trains.
Members of the Rail, Maritime and Transport union backed walkouts by 4-1 on a turnout of 76%, well above the 50% threshold in the Trade Union Act.
The company only took over from South West Trains in August, running services to and from London Waterloo.
It is now the fifth rail operator to be embroiled in a dispute over the role of guards and driver-only trains.
RMT members at Southern, Merseyrail, Arriva Rail North and Greater Anglia were said to be "solidly supporting" a 24-hour strike on Tuesday over the same issue, with another stoppage due on Thursday.
Picket lines were mounted outside stations, including in Manchester, where the Conservative Party conference is being held.
The new ballot result followed claims by the RMT that SWR franchise holder First/MTR had failed to give "cast-iron assurances" over the future of guards on its trains.
Union general secretary Mick Cash said: " RMT has given repeated opportunities for First/MTR to give us the assurances we have sought over the future role of the guards on their trains throughout the length of the new South Western franchise.
"They have refused to give us those guarantees and it is that failure which left RMT with no option but to declare a formal dispute with the company and to conduct a ballot for action.
"Our members have now voted overwhelmingly for both strike action and action short of a strike and that massive mandate will now be considered by RMT's executive, who will decide the next steps.
"This dispute can be easily resolved if the company are prepared to stick to existing agreements, give staff and passengers alike a guarantee of a second, safety-critical member of staff on all current services and an assurance that safety and access are the absolute priority."
The RMT has written to Prime Minister Theresa May, accusing her of "sabotaging" negotiations aimed at resolving the disputes.
The Merseyrail strike coincided with improvement work at Liverpool Lime Street station.
Merseyrail managing director Jan Chaudhry-van der Velde said: "At a time when passenger journeys are temporarily restricted due to these important investment works, the RMT has now targeted these as an opportunity to double up the inconvenience for passengers by staging two further strikes.
"So while the industry and key players in the city region work hard to improve the railway, the RMT seems to be doing everything in its power to stop it."
Greater Anglia is planning to run a full service on the strike days and Southern said there will be a normal service on most of its routes.
Arriva Rail North aims to run around 1,200 services across the North - 46% of its normal timetable - on both days between 7am and 7pm.
Most Merseyrail services will run between 7am and 7pm, with a break during the middle of the day, and some stations will be closed.
Paul Plummer, chief executive of the Rail Delivery Group, on behalf of train operators, said: "Passengers , communities and Britain want rail companies working together to deliver a long-term plan to improve services, not needless and opportunistic attempts to disrupt lives and the economy as we have seen across the North today.
"The RMT leadership need to work in the interests of the nation and resolve these disputes."
A South Western Railway spokesman said: "We are disappointed that some of our staff have chosen to vote for industrial action.
"None of us want to be in this position and any potential action will cause unnecessary disruption for the customers we serve.
"We have been clear that we plan to retain a second person on our services, as we know that our passengers value the assurance and assistance a second member of on-board staff provides.
"That's why, during our franchise, we will increase the numbers of drivers and guards so we can deliver more services for passengers.
"We aim to deliver improvements for passengers throughout the SWR franchise including new trains, quicker journeys, and earlier and later services.
"Today's announcement undermines those efforts. We urge the RMT to avoid premature strike action and work with us to deliver for our customers, and we look forward to meeting them again to discuss our future plans on October 5 as already arranged."
Rail minister Paul Maynard said: "The RMT should stop using passengers as pawns in their political game, call off strike action and hold constructive talks.
"Passengers will face needless delays, even though South Western Railway have been clear no one will lose their job.
"They plan to keep a second member of staff on board their trains and hire more drivers and guards.
"The RMT's dispute is not about safety, as the independent regulator has ruled that driver-controlled trains are safe."
The ballot result was announced hours after a second points failure in two days affected SWR services to and from London Waterloo.
A Network Rail spokesman said: "We sincerely apologise to passengers for the disruption to services today on the South Western Railway route.
"This has been caused by a points failure at Waterloo station closing platform one, and a signal failure at Crowthorne.
"We are working hard to resolve these issues and return services to normal as soon as possible.
"We thank passengers for their patience, and ask them to please check before they travel today."
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