Nine out of 11 academies, independent state schools backed by private sponsors, featured among the bottom 200 schools in England in last year's tests for 14-year-olds, the results of which were published this week.
The results, in maths, science and English, will raise further questions about the academy programme, which has already been attacked for diverting resources away from other schools and placing too much power in the hands of private sponsors.
Schools in the bottom 200 included the Business academy in Bexley, Kent, which was hailed as the future of secondary education when it was opened by Tony Blair in September 2003.
Capital City in Brent, north London, was the worst-performing academy, with just 28 per cent of pupils reaching the level expected of 14-year-olds in English, 35 per cent in maths and 23 per cent in science.
Nationally, 73 per cent of pupils reached level 5 - the standard expected of 14-year-olds - in English and maths, and 68 per cent in science. Scores were 2 per cent down on the target the Government set for all three subjects.
Unity City academy, in Middlesbrough, was rated 26th worst in the country using the alternative "value-added" measure, comparing pupil performance at KS3 with their ability when they joined the school.
The National Union of Teachers said the Government should now reassess its target of opening 200 academies by 2010. Steve Sinnott, NUT general secretary, said: "It is high time the Government reconsidered the strategy which is benefiting no one and using up a disproportionate amount of the funding that's needed for every child."
A Department for Education and Skills spokesman said: "Academies are a new type of school in some of the country's most economically and educationally disadvantaged areas, reinvigorating education and ... replacing schools which have failed their pupils for generations."
Philip O'Hear, principal of Capital City academy, said judgements based on the KS3 results were unfair because children sat the tests less than a year after the new-look school was opened. "We should be judged in three or four years' time," he said.
Mr O'Hear said more than two-thirds of students taking KS3 tests had not sat the corresponding exams at KS2. Many only entered the country in recent years or were absent from school at the time.
The poor performance of academies comes amid continuing criticism of the KS3 league tables, which were published for only the second time this week.
Publication of the tables was delayed after hundreds of schools complained about grades awarded to pupils in the English test. An official inquiry led to the resignation of the head of the National Assessment Agency in November.
The Association of Teachers and Lecturers is likely to call for KS3 tests to be scrapped at its annual conference on Wednesday.
The tables this year ranked Kendrick girls' grammar, Reading, as the top-performing school, while Dr Challoner's grammar, Amersham, was named as the best school on the value-added scale. In all, 31 of the 32 schools adding the most value between KS2 and KS3 were grammars.
How LEAs did at KS3