Ministers are facing fresh calls for an urgent review of this year's GCSEs amid continued concerns about English results.
In an open letter to Education Secretary Michael Gove and the exams regulator Ofqual, the National Association of Head Teachers (NAHT) claimed it was apparent that grade boundaries in English had been "significantly altered" during the year, and warned this could have "serious consequences" for pupils.
The union said it had been "inundated" with calls from schools about the situation, and urged Mr Gove to establish an independent inquiry.
National GCSE results for England, Wales and Northern Ireland, published on Thursday, revealed 69.4% of all GCSE exams were given at least a C grade - down 0.4 percentage points on last summer. It is the first time the A*-C pass rate has fallen in the 24-year history of GCSEs.
As the figures were published, angry headteachers began calls for an inquiry into English results, claiming that exam boards had raised grade boundaries in the subject halfway through the year amid fears that too many children were going to get a C.
A number of schools suggested that grade boundaries in this summer's English exams had been substantially increased, with many reporting a drop in the number of children scoring a C or above in the key subject.
In his letter, NAHT general secretary Russell Hobby said: "It has become apparent that grade boundaries were significantly altered between the January and June examinations series and the consequence of this has been that the overall requirement for attaining a C grade increased by 10 marks between January and June.
"NAHT believes that this is an iniquitous and unfair state of affairs, discriminating against those pupils whose schools took the decision to enter them in June. The decision will have serious consequences for those pupils adversely affected. In many cases, this may well prevent them from taking up opportunities to pursue A-level courses. This situation offends natural justice and is, quite simply, unfair."
The Academies Enterprise Trust (AET), which runs 29 academies, has asked schools to provide information about the backgrounds of pupils who they believe missed grades in English this year, the Times Educational Supplement (TES) reported. AET chief executive David Triggs said: "We want to find out whether this has actually affected the very pupils that the Government are trying to raise standards for. We want them treated fairly."
Mr Gove has refuted suggestions that there had been political interference in GCSE results, insisting that any changes in grades were the result of "independent judgments made by exam boards entirely free from any political pressure".