Worldwide, geomorphological classifications of estuaries are often used to guide the design of monitoring programs and management strategies. However, if classifications do not reflect biotic patterns, the effectiveness of monitoring and management is potentially reduced. In this study, we consider the effectiveness of one classification scheme in describing biotic patterns by examining and comparing spatial variation of macrofaunal assemblages and their relationship with the environment in 12 estuaries of 2 geomorphological types (mesotidal river dominated and permanently open barrier estuaries). Estuaries were sampled at three locations (upper, mid and lower) for macroinvertebrates and environmental characteristics. Differences in macrofaunal assemblages occurred between the estuary types at the lower and mid locations, but not the upper. Similarities in the upper locations were related to sediment, whereas at the mid and lower locations differences were linked to salinity, dissolved oxygen concentrations and seagrass area. Within estuaries, location effects were definitive and unique within each estuary type, correlating to changes in sediment particle size, nitrogen concentration, microphytobenthos and percentage organic carbon. These results suggest that estuarine classification based on physical attributes alone does have the potential to capture important biological attributes if the biological scales of variability within these systems are well understood.