Climate change has been shown to have a substantial impact on agriculture and high temperatures and heat stress are known to have many negative effects on the vegetative and reproductive phases of plants. In a previous study, we addressed the effects of high temperature environments on olive oil yield and quality, by comparing the fruit development and oil accumulation and quality of five olive cultivars placed in high temperature and moderate temperature environments. The aim of the current study was to explore the molecular mechanism resulting in the negative effect of a high temperature environment on oil quantity and quality. We analyzed the transcriptome of two extreme cultivars, ‘Barnea’, which is tolerant to high temperatures in regard to quantity of oil production, but sensitive regarding its quality, and ‘Souri’, which is heat sensitive regarding quantity of oil produced, but relatively tolerant regarding its quality. Transcriptome analyses have been carried out at three different time points during fruit development, focusing on the genes involved in the oil biosynthesis pathway. We found that heat-shock protein expression was induced by the high temperature environment, but the degree of induction was cultivar dependent. The ‘Barnea’ cultivar, whose oil production showed greater tolerance to high temperatures, exhibited a larger degree of induction than the heat sensitive ‘Souri’. On the other hand, many genes involved in olive oil biosynthesis were found to be repressed as a response to high temperatures. OePDCT as well as OeFAD2 genes showed cultivar dependent expression patterns according to their heat tolerance characteristics. The transcription factors OeDof4.3, OeWRI1.1, OeDof4.4 and OeWRI1.2 were identified as key factors in regulating the oil biosynthesis pathway in response to heat stress, based on their co-expression characteristics with other genes involved in this pathway. Our results may contribute to identifying or developing a more heat tolerant cultivar, which will be able to produce high yield and quality oil in a future characterized by global warming.