The hellbender, Cryptobranchus alleganiensis, is North America’s only giant salamander and is endemic to the eastern United States. Like many species of amphibians worldwide, hellbender populations are declining at alarming rates. Management efforts are essential to the continued survival of the hellbender. The Buffalo Zoo is raising hellbenders collected from the wild by the New York State Department of Environmental Conservation. These animals will be released back into the wild in an attempt to increase the overall population size. In order to understand the impact these animals will have on extant populations, both the genetic structure of the wild populations and the genetic composition of the released animals must be determined. Unfortunately, little is known about the genetic diversity and structure of hellbenders in New York. Therefore, this study used nine microsatellite loci to assess the genetic relationships of over 200 hellbenders from various locations throughout the Allegheny River drainage of New York and Pennsylvania and of the captive-raised individuals from the Buffalo Zoo. Based on previous research, hellbenders from different drainages differ strongly, while those from the same drainage are genetically similar. However, we found significant genetic differences between Allegheny tributaries. These genetic differences have implications for management as releasing captive-raised animals may alter the genetic structure of subpopulations.