The Canterbury club are now seven points behind South Group leaders Essex with one match remaining in the competition
Kent have been hit with a one-point deduction for a slow over-rate during the Bob Willis Trophy win over Surrey last month.
The penalty will take effect immediately, meaning the Canterbury club have been reduced to 63 points in the South Group.
They are now seven points behind leaders Essex heading into the final round of matches but remain in second place.
Only the top two ranked sides from the winners of the three regional groups will qualify for the Lord's final later this month.
The club have confirmed they initially appealed the judgment, which they claim came nearly 48 hours after the match concluded, and have criticised the ECB for the manner in which the episode was dealt with.
A statement added: "Whilst ECB acknowledged that communication was not effected as timely as it should have been, ECB have taken the view that there is nothing in the prevailing circumstances that would make the application of the penalty unreasonable."
Darren Stevens took 4-41 as Kent won by 17 runs on the fourth evening at The Kia Oval, as Surrey fell short of their target of 192 to win.
They face Hampshire in their final red-ball match, while pace-setters and last season's county champions Essex host Middlesex.
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