Summary The effect of high temperature stress (27ºC or 37ºC for 24 h) on total gene expression profiles in the annual-fruiting raspberry (Rubus idaeus L.) cultivars ‘Autumn Bliss’, ‘Autumn Treasure’, ‘Erika’, and ‘Polka’ were evaluated at the floral initiation stage using a customised Rubus microarray. Significantly affected genes were obtained by pairwise t-tests using ‘volcano plots’ for each cultivar × treatment. A 10ºC elevation in temperature altered levels of expression, in at least one cultivar, of 644 differentially expressed genes in total, with ‘Erika’ and ‘Autumn Treasure’ showing elevated expression of 38 genes compared to ‘Autumn Bliss’ and ‘Polka’. We identified 12 common candidate genes that were modulated differentially in ‘Autumn Bliss’ and ‘Erika’ at 37ºC compared to 27ºC. In addition, two aquaporin genes (PIP1 and TIP2) were down-regulated in ‘Autumn Bliss’, but up-regulated in ‘Autumn Treasure’, ‘Polka’, and ‘Erika’ at 37ºC. Other down-regulated genes from the list of 38 genes included those encoding major latex-like proteins, plasma membrane proteins, cysteine rich proteins, and other stress-related proteins. Validation by real-time quantitative RT-PCR (RT-qPCR) indicated subtle changes in differential gene expression, suggesting a mild response to heat stress. This study used molecular tools to increase our understanding of, and to identify candidate genes involved in, the heat stress response of four annual-fruiting raspberry cultivars.