The trio staged the show to an empty venue as fans tuned in from home due to the ongoing coronavirus lockdown guidelines.
The three stars' set ended with a cover of Diffie's John Deere Green.
"We lost a family member and peer and amazing man, artist, singer when Joe Diffie passed away...," Clark said. "I'm wearing my John Deere T-shirt here tonight in honour of Joe."
Diffie died on 29 March, after contracting the coronavirus. He was 61.
The Grand Ole Opry has been running its Saturday night performances in an empty Opry house since 14 March, in response to the coronavirus pandemic.
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