A total of 996 players and staff were tested between Tuesday May 19 and Friday May 22, with two employees from two clubs showing positive results.
Premier League clubs were allowed to return to small group training this week following the first round of coronavirus tests last weekend, of which six players and staff tested positive, three from Watford.
The six players to have tested positive in the first round are continuing to self-isolate and were not included in the second round of testing.
A 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.
"For the second round of testing, the number of tests available to each club was increased from 40 to 50.
"Previously, 748 players and club staff were tested for COVID-19 on 17-18 May, with six testing positive from three clubs.
"The six who tested positive in the first round of testing are not included in the figures for 19-22 May, as they are still within their seven-day self-isolation period.
"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."