Conserve the Chilterns and Countryside (CCC) has written a ten-page letter voicing its concerns.
The group, which includes residents and landowners, refers to the Aarhus Convention on public consultation, strategic environment assessment and the government's statutory duty to protect areas of outstanding natural beauty (AONBs).
SJ Berwin head of planning and environment Simon Ricketts said: "CCC does not object to HS2 on principle but does not understand why it has to bisect the Chilterns AONB at its widest point."
Meanwhile, the National Trust has spoken out against the proposed route because it would cut across its Hartwell House estate in the Vale of Aylesbury. The trust has also raised the need for a thorough consultation with the government.
"We hope to see a robust environmental assessment, a strong business case and a transparent consideration of alternative routes," declared the trust's assistant director of operations Helen Tuffs. The government plans to launch a consultation in the new year and publish a response in December 2011.
Campaign to Protect Rural England senior transport campaigner Ralph Smyth argued: "The controversy that these proposals are generating could derail the government's welcome vision to make rail the long-distance travel mode of choice."