Since April 2000, NOAA’s National Centers for Coastal Ocean Science (NCCOS) and the Office of National Marine Sanctuaries (ONMS) have been engaged in a collaborative partnership to augment the management of National Marine Sanctuaries (NMS) through increased scientific understanding of sanctuary sites (ONMS/NCCOS 2004). NCCOS scientists have been working with Gray’s Reef NMS (GRNMS) to assess the status of ecological condition and stressor impacts throughout the sanctuary and surrounding shelf areas, with a major focus on the soft-bottom benthos and sediment quality. Such activities are important to fulfill key research and monitoring goals for GRNMS in supporting scientific research and long-term monitoring to enhance the understanding of the Sanctuary environment and to improve management decision-making (NOAA 2014). An initial characterization of GRNMS was conducted in spring 2000 to evaluate the condition and distribution of benthic macroinfauna, including sediment-associated stressors, to provide a quantitative benchmark for tracking any future changes due to natural or human disturbances. The results of the spring 2000 survey suggested that soft-bottom habitats associated with GRNMS support highly diverse infaunal assemblages, thereby challenging a commonly-held assumption that these relatively featureless expanses of substrate surrounding “live bottom” habitat are biological voids (Cooksey et al 2004, Hyland et al. 2006). Levels of man-made, chemical contaminants were found at low, but detectable, concentrations in sediments and edible tissues of target species (black sea bass, turkey wing arks), suggesting background conditions while highlighting the need for future monitoring to track potential changes in levels of these substances. A follow-up survey in spring 2005 (Balthis et al. 2007) found similarly low levels of contaminants in sediments and fish and shellfish tissues, with measured concentrations being well below corresponding sediment quality and human health guidelines. Similar patterns in species richness and diversity of softbottom benthic assemblages also were observed in 2000 and 2005. This report presents the results of a follow-up survey, conducted in summer 2012/spring 2013, which provides additional data points for the long-term monitoring record as a basis for tracking status and trends in sanctuary conditions and supports important management products such as the GRNMS management plan (NOAA 2014) and GRNMS Condition Reports (ONMS 2008).