Kyrgios' absence was revealed when the US Tennis Association released the draw for the controversially remodelled event, which also confirmed Alex de Minaur's plea to be included with British fiancee Katie Boulter had been rejected.
Kyrgios has been troubled by injuries for three years and recently posted his knee was "cooked".
Australian world No.8 De MInaur, who had asked for a wildcard "pretty pretty please" via social media, is the highest-ranked man not in the event.
This was despite several other withdrawals, which left new pairing Jessica Pegula and Jack Draper as the No.1 seeds after both their planned partners pulled out.
British men's world No.5 Draper was due to play with Paula Badosa and women's world No. 4 Pegula with fellow American Tommy Paul.
There could yet be further withdrawals. The event begins Tuesday (local time) with the first round and quarter-finals being played then.
Yet Carlos Alcaraz plays Jannik Sinner in the final of the Cincinnati Open in Ohio on Monday. They are due to partner, respectively, Emma Raducanu and, in another new pairing, Katerina Siniakova. The Czech women's doubles No.2 has replaced Emma Navarro as partner of the men's singles world No.1.
French veteran Gael Monfils, who partnered Kyrgios in the DC Open in Washington recently, has replaced him as Osaka's partner.
Another eye-catching duo are 45-year-old Venus Williams and fellow American Reilly Opelka.
They are among eight wildcards with the other eight pairings selected on the basis of combined singles rankings - thus Draper and Pegula as top seeds.
The qualification policy is just one reason why the event, designed to attract attention of fans, sponsors, television and social media, is controversial.
Prize money has been increased five-fold to $US 1 million ($A1.5m) but only four games will be required to win a set in matches before Wednesday's final, with no-advantage scoring (four points will be needed to win a game) and 10-point match tie-breaks instead of a third set throughout.
Last year's US Open mixed doubles champions, Sara Errani and Andrea Vavassori have dismissed the new format as a "pseudo-exhibition focused only on entertainment and show" that would shut out true doubles players.
Having been given a wildcard they will have the chance to make their case for doubles specialists on court with their first match against No.2 seeds Elena Rybakina and Taylor Fritz.
US OPEN MIXED DOUBLES DRAW
Jessica Pegula & Jack Draper (1) v Emma Raducanu & Carlos Alcaraz
Olga Danilovic & Novak Djokovic v Mirra Andreeva & Daniil Medvedev
Iga Swiatek & Casper Ruud (3) v Madison Keys & Frances Tiafoe
Naomi Osaka & Gael Monfils v Caty McNally & Lorenzo Musetti
Katerina Siniakova & Jannik Sinner v Belinda Bencic & Alexander Zverev
Taylor Townsend & Ben Shelton v Amanda Anisimova & Holger Rune (4)
Venus Williams & Reilly Opelka v Katarina Muchova & Andrey Rublev
Sara Errani & Andrea Vavassori v Elena Rybakina & Taylor Fritz (2)
(seeds in brackets)
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