Thirlmere Lakes, in the Greater Blue Mountains World Heritage Area, consist of a group of five waterways (Lake Gandangarra, Lake Werri Berri, Lake Couridjah, Lake Baraba and Lake Nerrigorang). Water levels in Thirlmere Lakes have fluctuated over time, but there has been a recent decline that is of significant concern to the local community. There is still much that is unknown about Thirlmere Lakes including their geology, geomorphology, hydrogeology and hydrology; without this information the exact cause of decreasing water levels in the lakes remains a mystery and it is difficult to determine what the primary drivers of surface water loss are. In this study, we examined the species composition of zooplankton (rotifers, cladocerans and copepods) and physico-chemical conditions (temperature, dissolved oxygen, pH, conductivity, turbidity and nutrients) of Lake Werri Berri and Lake Nerrigorang once a month for a year, including hydrogen and oxygen isotopes (δ2H and δ18O) in Lake Werri Berri. We found 68 taxa of zooplankton including the first record of the rotifer Rousseletia corniculata Harring, 1913 for New South Wales. The two lakes shared 30 % of the taxa. There was a marked difference in water quality between two lakes, with more variable conditions in Nerrigorang than Werri Berri. Although the two lakes are closely situated, and thought to potentially be connected at high water levels, they show distinct ecological characters.