Ozark hellbenders (Cryptobranchus alleganiensis bishopi) are large aquatic salamanders found in flowing waters. The abundance of this species is thought to have declined over the long term. What had been the most abundant population, in Spring River, Arkansas, appears to have declined precipitously in the last decade. The possibility of supplementing the population through captive propagation has been suggested, raising concerns about genetic issues. Random amplified polymorphic DNA (RAPD) was evaluated for assessing genetic variability among and within populations of Ozark hellbenders from Spring River (Ark.), Eleven Point River (Ark.), and North Fork White River (Mo.). Six primers were tested, and all generated reproducible RAPD profiles. Forty RAPD bands were generated of which 20% exhibited within-population variation and an additional 5% exhibited between-population variation. Analysis using primer OpB12 produced 2 fixed markers that distinguished hellbenders from the Spring River and North Fork White River from those in Eleven Point River. The distinct RAPD profiles of different populations suggest a lack of gene flow and possible effects of inbreeding and random genetic drift. This is in agreement with previous studies that examined isozymes and mitochondrial DNA (mtDNA). Previous isozyme data, which is based on actual proteins and phenotypic variation indicate that Spring River and White River are identical populations and North Fork White River could be used for restoration efforts. RAPD and mtDNA data combined, which are based on non-coding regions of the genome indicate that the 3 populations examined are uniquely different from each other and should not be mixed. No data on the quantitative genetic variation is available. In light of the population structure of these hellbenders, apparent strict reproductive isolation and the development of highly homozygous lines, and possible habitat degradation, intentional crossing of those populations could actually increase their fitness and viability. To provide genetic information useful for restoration efforts, further studies are needed to assess the population structure and geographical distribution of various genotypes.