Cryptobranchus alleganiensis alleganiensis (Eastern Hellbender) is an environmentally sensitive species that has experienced range-wide population declines. Diurnal rock-turning surveys were conducted in southern WV during the summer and fall of 2006 to assess the species' population status in this area and to examine the relationship between stream physico-chemical characteristics and site occupancy. Survey results suggest that Eastern Hellbender populations are rare in southern WV, with Eastern Hellbender present at only ≈15% of all sites surveyed and only ≈20% of sites where they have been documented historically. Logistic regression models showed that presence of increased gravel substrate and specific conductivity reduced the probability of site occupancy by Eastern Hellbenders. It is not clear why a higher proportion of gravel substrate negatively affected site occupancy, because gravel should benefit Eastern Hellbender populations by providing larval habitat and habitat for prey species. The effect of specific conductivity may indicate a negative impact of watershed disturbance on populations. This explanation is supported by a principal component analysis of habitat characteristics followed by logistic regression, which demonstrated that sites with habitat characteristics indicative of more degraded sites (e.g., higher specific conductivity) decreased the probability of a site being occupied by Eastern Hellbenders. The results of our study suggest that Eastern Hellbender populations may be severely threatened in southern WV and that site occupancy by Eastern Hellbenders is related to both the physical nature of stream substrate and to water quality characteristics. Furthermore, this study indicates a need for research investigating the potential for human land-use to adversely affect Eastern Hellbenders.