Failing to teach evolution by natural selection in science lessons could lead to new free schools losing their funding under government changes.
The new rules state that from 2013, all free schools in England must teach evolution as a "comprehensive and coherent scientific theory".
The move follows scientists' concerns that free schools run by creationists might avoid teaching evolution.
Sir Paul Nurse, president of the Royal Society, said he was "delighted".
Sir Paul told the previous rules on free schools and the teaching of evolution versus creationism had been "not tight enough".'Creationist myth'
He said that although the previous rules had confined creationism to religious education lessons, "the Royal Society identified a potential issue that schools could have avoided teaching evolution by natural selection in science lessons or dealt with it in a such a perfunctory way, that the main experience for students was the creationist myth".
So far 79 free schools have opened in England with 118 more due to open in 2013 and beyond. They are funded directly by central government but unlike other state-funded schools are run by groups of parents, teachers, charities and religious groups and do not have to abide by the national curriculum.
The new rules mean if a free school is found to be acting in breach of its funding agreement - for example, teaching creationism as a scientific fact or not teaching evolution - the Department for Education will take "swift action which could result in the termination of that funding agreement".
In a letter to the Royal Society, the Schools Minister, Lord Hill, said: "While we have always been clear that we expect to see evolution included in schools' science curricula, this new clause will provide more explicit reassurance that free schools will have to meet that expectation."
Sir Paul Nurse said: "The new clause in the funding agreement should ensure that all pupils at free schools have the opportunity to learn about evolution as an extensively evidenced theory and one of the most fundamentally important tenets of modern biology.
A spokesman for the Department for Education said that the new clause would apply to the Grindon Hall Christian school in Sunderland and two others that this year became the focus of concerns about the teaching of creationism in free schools.
Grindon Hall, which was independent, reopened as a free school in September. The two others approved by ministers are not due to open until 2013.
In July the principal of Grindon Hall said that creationism would never be taught in science lessons.'Additional safeguards'
Rachel Wolf, director of the New Schools Network, which provides advice and support for groups who want to set up free schools, welcomed the funding agreement changes but said that the existing rules meant free schools already had to teach evolution in science lessons.
"To my knowledge free schools have always had to teach evolution in science, but it is great that the government has reaffirmed its commitment to this," she said.
Andrew Copson, chief executive of the British Humanist Association, organisers of a Teach Evolution not Creationism campaign, said: "A requirement to teach evolution in free schools is an excellent additional safeguard against state-funded creationist schools and must be welcomed.
"However, we continue to be concerned about the three free schools recently approved which are supportive of teaching creationism as science and which we must worry will continue to find ways to circumvent a ban in practice."
Dr Berry Billingsley who leads a Reading University project on how secondary schools handle questions that bridge science and religion cautioned against an oversimplified debate.
"Evolution is a fantastic theory and explains so much about how humans come to be here. It is backed up by evidence and supported by the vast majority of scientists in the biological sciences. Many of those scientists also believe that the Universe is here because of God.
"The importance of studying evolution is indeed the first thing to be said but children also need opportunities somewhere in the timetable to explore the 'Big Questions', which our research shows they want to consider and it is often the science lesson that stirs up those questions."
Paul Bate, of the European Educators Christian Association, agreed schools should teach a broad and balanced curriculum: "Science and religion need each other in this debate. Albert Einstein, one of the greatest scientists of all time said, 'Science without religion is lame, religion without science is blind.'"