I'm looking at a trailer to buy that roll on tires that are 24.5, my tractor tires will be be 22.5. Will the difference in size present any problems?

The only problem, i can see, is that you won't be able to put your partially worn steers on a trailer.
Otherwise it doesn't matter!

and if you carry a spare, it will wont fit both.

You could always switch your trailer down to 22.5 also that would lower the height also. That way you could also use your old steers on the trailer. A 22.5 carries the same weight as a 24.5 so no penalty there plus a 22.5 tire an wheel assembly is about 100 lbs lighter per side less steel and rubber so more cargo room.

There's good news- With bigger tires on yout trailer, your rig will always be heading downhill!! That's gotta' help your fuel mileage...