Thirty-five tree species, represented by 240 individuals, were climbed during two three-week periods in June, July, and August of calendar years 2000 and 2001 in the Great Smoky Mountains National Park. The double rope climbing technique was used because the climbers could advance to higher levels in the tree canopy. Maximum-sized mature trees in old growth park areas were sampled up to 40 m. Bark samples were cultured in moist chamber cultures. Ninety-five myxomycete species were obtained from the tree canopy mostly from the following tree species: Fraxinus americana (white ash), Quercus alba (white oak), Liriodendron tulipifera (yellow poplar), Acer rubrum (red maple), and Pinus strobus (white pine). We report here 52 new records from the tree canopy, 7 from ground sites, bringing to 212 the number of myxomycete species known from the Park. Comatricha penicillata is recorded for the first time outside of the type locality in Japan. This is the first upper tree canopy list of myxomycete species from the Park. The Myxomycetes represent the only group of cryptogams with species only known from the tree canopy.