Tests were carried out at training grounds across the country on Tuesday, Thursday and Friday and the results were sent out on Saturday.
The two positive cases are from two clubs and are seperate from the six cases found in the first round of testing.
A Premier League statement read: ‘The Premier League can today confirm that on Tuesday 19 May, Thursday 21 May and Friday 22 May, 996 players and club staff were tested for COVID-19. Of these, two have tested positive from two clubs.
‘Players or club staff who have tested positive will now self-isolate for a period of seven days.
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‘The Premier League is providing this aggregated information for the purposes of competition integrity and transparency.
‘No specific details as to clubs or individuals will be provided by the League and results will be made public after each round of testing.’
No names or clubs have been confirmed after the results.
After the first round of testing, Watford’s Adrian Mariappa, two members of the clubs staff and Burnley assistant manager Ian Woan were confirmed as among the six positive tests.
Players in the Premier League resumed training on Tuesday and there is a target of 12 June as a restart date.
Premier League chief executive Richard Masters is still hopeful of achieving that aim, speaking on Friday he told BBC Sport: ‘There is some momentum. We’ve taken the first step.
‘It’s great for everybody, including the fans, to see our players back on the training ground.’
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