Added a spoiler warning to the thread title.

Biggest betrayal was Bungie not being loyal to MS since they became what they are today thanks to them!

Metal Gear Solid Namoi - seriously injecting Snake with a dangerous virus that later threatens all of humanity is not cool. Also Revolver Ocelot can't decide what side he is on. Also ironic how she was ratted out by Liquid Snake who also ends up betraying you.

This one's more deception than outright betrayal, but 999: Ace, aka Gentarou Hongou. Went from chill old dude to my most reviled video game character ever once he revealed his true nature.

Mario ditching Yoshi in Super Mario World

Then Phillips stick the knife into Nintendo, over motion control patents, its a merry go round of betrayal.

Kitler53's betrayal to Nintendo.

On one of the Hyrule Warriors Adventure Maps, I was fighting two Gohmas as Lana...when mere seconds into the battle, I discover that Ghirahim betrayed me. It shook me to the core. I trusted him so much!

Though seriously, Dickson probably takes it for me. 

Arc Rise Fantasia - Almost the entire party ditches you about ten hours in (including two childhood friends!), then actively try to defeat you for the rest of the game. Hardest left turn I've seen a JRPG take in a long time.

Really?