Boris Johnson's father said he was "disappointed" not to have been selected as a possible successor to his son as MP for Henley.
Stanley Johnson, who is based in Devon, said it would be "superb" to represent the constituency in Oxfordshire.
But the Conservatives have instead picked three local candidates as possible contenders.
A date for the by-election, which has come about by Boris Johnson becoming London's mayor, is yet to be set.
There have been reports the party could capitalise on its by-election victory in Crewe and Nantwich by setting a mid-summer date to elect a new MP for Henley.
'Local candidate'
But any contest will not feature Stanley Johnson, who said: "Of course I am very disappointed but I certainly understand and accept the reasons.
"The local Conservative association has decided in this instance to restrict the short-list to local candidates.
"I am sure whoever is selected by the members will do a superb job and will win a convincing victory."
Stanley Johnson has previously worked in the European Commission.
The three Tory candidates are local councillors John Howell, Ann Ducker and John Cotton.
John Walsh, chairman of the South Oxfordshire Conservative Association, said: "Where we have not chosen a local candidate the Liberal Democrats very much pick up on that and criticise it significantly.
"I think it is designed this time that nothing goes wrong. The primary importance is to make sure our electors turn out."
Labour's Richard McKenzie, the Liberal Democrats' Stephen Kearney and the Green Party's Mark Stevenson have already been selected to fight a by-election. The UK Independence Party has selected Chris Adams.
Boris Johnson succeeded former Deputy Prime Minister Michael Heseltine as MP for Henley at the 2001 general election with a majority of 8,458.
He was re-elected in 2005 with an improved majority of 12,793 and a 53% share of the vote.
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