The Eastern Cape Karoo region is semi-arid with highly variable rainfall. This variability in rainfall sustains a mosaic of surface freshwater bodies that range from permanently to temporarily inundated. These waterbodies provide habitats for diverse invertebrate assemblages. The imminent hydraulic fracturing for shale gas has a potential to modify the water regime, with particular risk of salinisation. Accumulation of salts in freshwater wetlands results in loss of biodiversity, as invertebrate species shift from salt intolerant to salt tolerant species. This study therefore aims to expand on existing knowledge and provide new information on the distribution, diversity and structure of macroinvertebrate assemblages associated with various freshwater bodies in the region prior to shale gas exploration. Limnological and ecological aspects of thirty-three waterbodies (rivers, dams and depression wetlands) were investigated between November 2014 and March 2016. An experimental study on the effects of salinity on hatching success of branchiopod resting eggs was also included in the research. Rivers were characterised by high conductivity and depression wetlands by high turbidity, while dams had relatively higher pH than the other two waterbody types. In terms of global phosphorus interpretation guidelines, the results indicate that freshwater systems in the study region are predominantly eutrophic, indicating that agricultural run-off, particularly from livestock dung (goats, cattle and sheep), is an important source of phosphorus in the freshwater systems studied. Our results revealed new distribution records for branchiopod crustaceans in the Eastern Cape region, including the first record of Laevicaudata. Results showed that the sampled variables were unable to explain the variation in physicochemistry and invertebrate assemblage of several sites. Waterbody type, whether a depression wetland or a river, was the only factor that consistently showed an effect on the composition of both physicochemical data and invertebrate data. Depression wetlands ranged from completely bare to being extensively covered by macrophytes. Therefore, the effect of macrophyte cover in structuring macroinvertebrate assemblages was the focus of further investigation. The results indicated that the macrophyte cover gradient had little influence on the structure of the invertebrate assemblages in the depression wetlands, while only the presence/absence of vegetation significantly influenced the structure of the invertebrate assemblages in these systems. Surface area, dissolved inorganic nitrogen and pelagic chlorophyll-a were the environmental variables that best explained the variation in the macroinvertebrate assemblages among the sites. However, the differences in macroinvertebrate richness, abundance and distribution patterns among sites were only weakly influenced by local and regional environmental factors. These findings suggest that invertebrate in temporary wetland systems are adapted to the highly variable nature of temporary habitats, thus the influence of local variables is negligible. Results of the experimental study, on the effect of salinity on hatching success of branchiopod resting eggs, revealed that hatchling abundance and diversity of large branchiopods was significantly reduced at salinities of 2.5 g L−1 and above. Salt-tolerant taxa such as Copepoda and Ostracoda were the only ones to emerge in the highest salinity of 10 g L−1. Thus, should the region continue to experience increasing aridity and possible shale gas development, which all aggravate the salinisation problem, severe loss of branchiopod diversity (Anostraca, Laevicaudata, Spinicaudata and Notostraca) is likely to occur. This may lead to considerable decline in invertebrate diversity in the region, with cascading effects on food webs and ecosystem functions. The findings of this study can potentially be used in comparative studies on wetland invertebrate ecology in other semi-arid regions and in the formulation of policy and strategies for biodiversity conservation.