...that the three "girls" were guys right from the moment she saw them? It kinda seemed she knew, but she just went along with it. But she WAS surprised when Jim tells her he's not really a girl though......
I think she knew, but she was just trying to use the situation to her advantage. Then when he came out with it she made a scene to try to make him look bad to Cates' character.
Yes, I think you are 100% right ArmandoSerrano, as it is obvious from the way she pushes Bubba into Betsy's room when she first sees them, ignores Roy, and takes Jim into hers, then begins to undress and has him message her, then reacts that way when he reveals this and it leads right out to the hall and "Cates'" return, when she says that to her. It is obvious, and is made clear earlier in the movie that she was trying to steal Jim from her, and get him for herself, and she saw this as a perfect opportunity.
Plus, you forgot to mention is that Betsy intentionally cranked up the heat just so that Jim could sweat a little. When he says it is getting hot in the room she raises the thermostat even more to make the room hotter. I do think she knew that it was the guys from the start but played as if she did not really know though.