The present study provides a detailed analysis of the factors influencing variation in cyanobacterial communities of a large shallow off-river drinking water reservoir on the east coast of Australia. Receiving multiple inflows from two unprotected mixed land-use catchments, the Grahamstown Reservoir is a model example of a reservoir which is highly vulnerable to adverse water quality issues, including phytoplankton blooms and the resulting filtration, toxin and taste and odour problems produced. The spatial and temporal distributions of cyanobacteria were assessed for a period of 3 years (January 2012–December 2014) based on samples collected from three monitoring stations within the reservoir. Relationships between cyanobacterial abundance and a range of environmental factors were evaluated by application of multivariate curve resolution–alternating least squares (MCR-ALS) analysis. Results of the analysis indicated that among the 22 physico-chemical variables and 14 cyanobacterial taxa measured, the vertical temperature gradient within the water column and nutrient availability were the most powerful explanatory factors for the observed temporal and spatial distribution patterns in the densities of cyanobacterial taxa. The abundance patterns of the dominant cyanobacterial taxa—Aphanocapsa, Aphanothece, Microcystis and Pseudanabaena—were strongly linked with rainfall and run-off patterns into the reservoir, while Coelosphaerium and Microcystis were the taxa most influenced by the apparent occurrence of thermal stratification. The findings demonstrate the capacity of rigorous multivariate data analysis to identify more subtle relationships between water quality variables, catchment factors and cyanobacterial growth in drinking water reservoirs.