HS2 risks dividing and destroying “huge swathes” of “irreplaceable” natural habitats, including 108 ancient woodlands, a report has warned.
The Wildlife Trust said the high-speed rail line linking London and northern England could wipe out rare species.
The organisation says if the project, which is currently on hold, goes ahead a “greener” approach will be needed.
HS2 Ltd said its railway will respect the environment through the creation of a “green corridor” along the route.
The government commissioned a review into HS2 in August and is set to decide in the coming weeks whether to proceed.
The Wildlife Trust said its report – which uses data from 14 local trusts affected by the plans – is the “most comprehensive” assessment of the environmental damage the high-speed rail line could cause.
It claims HS2 could have a significant impact on hundreds of nature reserves, sites of Special Scientific Interest and ancient woodlands.
The organisation fears rare species such as the Dingy Skipper Butterfly could become extinct in some local habitats.
Barn Owls and the endangered White Clawed Crayfish could also be impacted by the project, it added.
The Wildlife Trust called on the government to “stop and rethink”.
Nikki Williams, the Wildlife Trusts’ director of campaigns and policy, said: “HS2 will destroy precious carbon-capturing habitats if it’s allowed to continue in its current form.
“It will damage the very ecosystems that provide a natural solution to the climate emergency.”
She labelled HS2’s proposed measures “amateurish”.
Hilary McGrady, director general of National Trust, said HS2 Ltd has “a vital responsibility to lead by example” and “must not end up cutting corners at the expense of the environment”.
HS2 said it has carried out extensive work to relocate animals, such as the Great Crested Newt, away from sites and into newly-created habitats.
The company said the “green corridor” will deliver a railway that “respects” the natural environment.
According to the company’s website, 3.4 square miles (9 sq km) of new woodlands – made up of seven million trees and shrubs – will be created along the first phase of the project between Birmingham and London.
It claims the figure is more than double the amount affected by the project.
A further 1.5 square miles of wildlife habitat will be established along the route, HS2 adds.