I was interested in a hotel in Rome and found rates for half the price on Expedia, Travelocity, and Hotels.com. The rooms was described exactly the same on these agency sites as by the hotel. The hotel wasnted 459 Euro for a superior room and it was 254 Euro on the agency sites. I emailed the hotel and they said that if I purchased my room through one of the agencies, I would not get the same quality room as if I book directly with them. What is your opinion of this and has this been your experience when traveling?

I rarely book thru any site other than the hotel's, but have read at least two interviews with former hotel staff who confirm that discounted bookings are often given 'lesser' accommodations.
Could the hotel describe what the difference in accommodation would be? Smaller room, or shower only, or no view or...? It might be an acceptable tradeoff to you. Or it might be a case of renovated vs. old rooms.

What hotel is it? Did the hotel describe the differences between the two types of rooms in its email?

If the hotel isn't willing to commit to what they will offer you in each case, I think they are just trying to get the highest rate they can out of you with this unspecified threat. If both the agency site and the hotel site say "superior room", I think I would just skip this hotel altogether, based on that response.

Hotel Nazionale on Plazza Montecitorio.
I explained the difference in prices and their response was simply, they would not give us the same quality of room if we did not book with them. Leaves me feeling cold. Do not think I would want to stay there now with no further explanation. I was just curious if this is the common policy with hotels in Rome. If they allow so many rooms to be on the agency sites, then why cannot they honor securing you a comparable room?

Here's a recent SlowTrav review from that hotel where someone was disappointed with Hotel Nazionale on Plazza Montecitorio, which they got after being bumped from another hotel:
http://tinyurl.com/yywt6f

Note the reviewer doesn't feel it was worth 195 Euros per night (albeit in a low-season time). For 459 Euros per night, it would have to be pretty spectacular, and it doesn't seem to be a spectacular type of hotel.

I think it is a crap shoot with some hotels. But in your case, I would move on. That hotel may have had renovated and non-renovated rooms and the cheaper ones were probably unrenovated. Fair enough, but that should have been the explanation (if it was the case) not the bias toward direct booking. If the room described was superior, then it should have been superior.