I believe they knew. Here is a quote from the Pilot Colonel Tibbets: "When the bomb is dropped, Beser [the radio officer] will record your reactions. This record is for history, so watch your language."

Source: Hiroshima by Stephen Hoare. Probably in plenty of other places too, but that's where I read it.

Here are some other reasons I think they must have known (I can't remember where I learnt these, but it is likely to be the same book, or Hell in the Pacific by Jonathan Lewis & Ben Steele):

• They waited for a clear day and flew with some other planes, whose purpose was to observe.
• The bomb was in the plane and it weighed around 4 tons and they must have been aware it wasn't normal.

P.S. I expect someone better informed than myself is going to be able to tell you more directly, where I have only been able to speculate.

edit: clarity

The unit that flew the aircraft, the 509th Composite Group was specifically formed to train and develop methods for dropping atomic bombs. The aircraft were specially modified to carry the uniquely shaped bombs.

Col Tibbets was the 509th's commander and had been training with the unit since Dec. of 1944, and then was the pilot on the Hiroshima mission.