A change to the route was announced in July which would see the high speed service run through Aston and Bramley.
Protesters took to the streets of Bramley earlier over fears about 70 homes could be demolished.
A spokesman for HS2 said the line would "dramatically improve" journeys between the Midlands, the north of England and London.
Rotherham Council has opposed the plan and Rother Valley MP Kevin Barron said he had applied for a debate in the House of Commons about the proposal.
Jean Handley, who lives in Bramley, said: "People in the community are saying they don't want this.
"It's not just the fact it's going to run at the bottom of the housing estate, it's the disruption and all the traffic going through."
Fellow protester Sandra Hay added: "The train won't stop in Rotherham, it's no use to us."
The proposed new route would see the HS2 line passing between Rotherham and Doncaster.
Plans for a station in Meadowhall were scrapped in July in favour of a stop in Sheffield city centre.
A spokesperson for HS2 said: "We believe the new South Yorkshire route will be cheaper, easier to construct, provide better connectivity and affect fewer people than the Meadowhall option.
"We will continue to work with local authorities, businesses and people along the line of route to better understand their concerns."
The plans will be considered later in the year.
Campaigners have marched through a Rotherham village in a protest about HS2.