Spatial and temporal patterns of variability in ascidian assemblages were investigated on horizontal subtidal rocky reefs at Port Stephens, New South Wales (NSW). The study was designed to provide a baseline dataset on ascidian diversity and distribution patterns for an area destined to become a marine park (the Port Stephens–Great Lakes Marine Park: PSGLMP). Differences in ascidian assemblages between exposed oceanic island reefs and sheltered reefs within Port Stephens, and between two depth zones within each subtidal reef, were quantified using non-parametric multivariate techniques coupled with analysis of variance (ANOVA). Ascidian assemblages were highly variable between reef sites, reef exposures and particularly between depth zones within each reef surveyed. However, temporal variation was only observed for a few ascidian species. These highly variable spatial patterns in diversity indicate that numerous subtidal reefs may need to be protected within PSGLMP if the aim of the marine park is to adequately represent the entire array of marine biodiversity in the area.