One of the basic tenets of ecological niche theory is that closely-related, ecologically similar species do not coexist. When such species co-occur over large portions of their geographic ranges, they often are segregated by habitat. The objective of this study was to evaluate experimentally the roles of abiotic habitat characteristics and competition in the habitat segregation of two sister taxa of Hylid treefrogs. These species co-occur throughout the southeastern United States, but Hyla cinerea typically breeds in permanent ponds, while H. gratiosa breeds in temporary ponds. I conducted a reciprocal transplant experiment using enclosures in natural ponds to compare survival, larval period, and size at metamorphosis for both species in temporary and permanent ponds. While the overall survival of H. cinerea was higher than H. gratiosa and survival varied among localities, there were no significant distinctions in survival between temporary and permanent ponds. Hyla cinerea tadpoles had longer larval period and larger size at metamorphosis in temporary ponds relative to permanent ponds. Hyla gratiosa tadpoles were significantly larger at metamorphosis in temporary ponds than permanent ponds, and had longer larval periods when encountering only conspecifics than when raised with H. cinerea. The results of these experiments, in conjunction with other work, suggest that a combination of factors is likely responsible for the habitat segregation between these two species.