The 28-year-old took gold in Delhi four years ago but was unable to make it out of the heats at Hampden Park on Wednesday morning.
Greene, crowned world champion in 2011, has barely raced this year due to injury and that lack of fitness showed as he tailed off dramatically towards the end of the race.
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The Welshman began strongly enough but faded badly down the home straight, crossing the line fifth in 50.36 seconds - way off his personal best of 47.84 secs.
Greene said: "I knew I was playing catch-up from a few months ago.
"I didn't expect to be here but my mindset did shift a bit and I wanted to make the final but ran out of steam, sadly it wasn't to be.
"I was provisionally selected for the European Championships but I don't think I'll make the squad now.
"I'm not fit enough really. It has been frustrating but I'm happy to have given it a shot and the support I have had has been fantastic, so thank you."
Greene's absence is another blow for Wales, who lost 2010 bronze medallist Rhys Williams before the Games after he was charged with an anti-doping violation.
The only home nations representative in Thursday's final will be Richard Yates after the English runner qualified as a fastest loser in 49.80.
English duo Niall Flannery and Seb Rodger failed to progress from the 400m hurdles heats, as did Northern Ireland's Jason Harvey.
The home nations fared better in the 200m heats earlier in the morning, with four sprinters progressing to the semi-finals.
Northern Ireland's Leon Reid qualified as a fastest loser, while England pair Christopher Clarke and James Ellington made it after finishing second in their heats.
It was Danny Talbot who led the home nation charge, though, crossing the line as joint-second fastest qualifier in 20.56.
"I've had a really good couple of weeks of training, I feel good," the British champion said.
"I've been around my PB a lot this season and in different conditions, mainly into headwinds, so I feel if the conditions are good I should go a lot faster than my PB."
After news emerged that Scotland's Laura Muir, fresh from her disappointment in last night's 1,500m final, had withdrawn from the heats, another home favourite, Lynsey Sharp, helped put a smile back on the local's faces by qualifying from the first heat.
Fellow Scot Emily Dudgeon also made it through, while England's Jenny Meadows, Jessica Judd and Marilyn Okoro also progressed.
Elsewhere in the morning session, Shara Proctor made the women's long jump final with a leap of 6.51m, Wales' Brett Morse progressed to the discus final and Jessica Taylor remained third in the heptathlon standings.