Background Different locations and study periods were used in the assessment of the relationships between heatwaves and mortality. However, little is known about the comparability and consistency of the previous effect estimates in the literature. This study assessed the heatwave mortality relationship using different study periods in the three largest Australian cities (Brisbane, Melbourne and Sydney). Methods Daily data on climatic variables and mortality for the three cities were obtained from relevant government agencies between 1988 and 2011. A consistent definition of heatwaves was used for these cities. Poisson generalised additive model was fitted to assess the impact of heatwaves on mortality. Results Non-Accidental and circulatory mortality significantly increased during heatwaves across the three cities even with different heatwave definitions and study periods. Using the summer data resulted in the largest increase in effect estimates compared to those using the warm season or the whole year data. Conclusion The findings may have implications for developing standard approaches to evaluating the heatwave-mortality relationship and advancing heat health warning systems. It also provides an impetus to methodological advance for assessing climate change-related health consequences.