They have seven days to reply and a decision will then be made on whether to hit the club with a charge - but it does not look good for the Seasiders.
There is a feeling within the Premier League that it would set a dangerous precedent if they allowed Blackpool to go unpunished. And if they do act, it will put them on a collision course with Holloway who has twice said he will resign if the club are fined for his selections.
Plenty of explaining to do: Blackpool boss Holloway changed the whole team at Upton Park
After a 2-2 draw with Everton on Saturday, November 6, Holloway made 10 changes at Aston Villa on November 10 when Blackpool lost 3-2, and he then changed the entire starting line-up for Saturday's 0-0 draw at West Ham.
The Premier League waited to see the team at Upton Park before deciding to formally press ahead with an investigation when they will decide if the integrity of the competition has been damaged.
It was also noted that five Blackpool players were making their first starts of the season in the Premier League against Aston Villa and Ludovic Sylvestre was making only his second.
Holloway's post-match comments have also been taken into account.
'I've been working with these players for however many weeks and I said I was going to give them a chance,' he said, after losing to a last-minute goal at Villa.
'The right back cost me £1million, the goalkeeper was a Ghanaian international, the Israeli centre half is an international and the left back is an Australian international.'
All change: Keith Southern was part of a number of changes made by Blackpool boss Holloway against Aston Villa
If the Blackpool boss had then played some of the same players days later at West Ham, the Premier League may have taken his point, but he dropped them all and recalled those generally considered to form his strongest team.
'If the Premier League are looking for me to explain it, I can explain it every step of the way,' said Holloway, and it looks as though he will have to.
Wolves were hit with a £25,000 suspended fine last season when manager Mick McCarthy made 10 changes against Manchester United in an effort to keep his first team fresh for a key a relegation battle with Burnley five days later.
After losing 3-0 at Old Trafford, Wolves beat Burnley 2-0. Fulham, however, were not punished when Roy Hodgson made extensive changes to his team in the Premier League before important Europa League ties last season, even though West Ham lodged a complaint.
That followed six Fulham changes in the 2-0 defeat by Hull, a team who were locked in a relegation fight with the Hammers at the time.
Welcome back: David Vaughan returned to Blackpool's changed side against West Ham
When Manchester United fielded a reserve team against Hull on the final day of the 2008/09 season, three days before the Champions League final, they were told they would be charged if they did not win. Sir Alex Ferguson's men went on to clinch all three points with a 1-0 victory, before losing 2-0 to Barcelona.
There clearly is a subtle difference between using the resources of a squad and fielding a weakened team and naturally this policy favours wealthier clubs with better playing strength.
But the Premier League insist that all clubs have been made fully aware that their team selections must uphold the integrity of the competition.
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