No people were injured in the single vehicle accident, which occurred near Grangemockler.
The horses were Do As You Wish, a five-year-old mare who had run four times and was due to contest the Ardfinnan Maiden Hurdle; and Uffizy, another maiden five-year-old mare who had run six times and was due to run in the Slievenamon Beginners Chase.
They had been withdrawn earlier in the day, with transport difficulties noted as the official reason for their withdrawal.
Tony Mullins, a brother of champion trainer Willie Mullins, trains at Gowran in Co. Kilkenny.
On Tuesday night he tweeted “very upsetting day today for our yard but we will just have to dust ourselves off and start again. Horses killed in a road accident. No people injured.”
Investigations into the accident are continuing.