BACKGROUND: Head formation of broccoli (Brassica oleracea var. italica) is greatly reduced under high temperature (22 °C and 27 °C). Broccoli inbred lines that are capable of producing heads at high temperatures in summer are varieties that are unique to Taiwan. However, knowledge of the early-activated pathways of broccoli head formation under high temperature is limited. RESULTS: We compared heat-tolerant (HT) and heat-sensitive (HS) transcriptome of broccoli under different temperatures. Weighted gene correlation network analysis (WGCNA) revealed that genes involved in calcium signaling pathways, mitogen-activated protein kinase (MAPK) cascades, leucine-rich repeat receptor-like kinases (LRR-RLKs), and genes coding for heat-shock proteins and reactive oxygen species homeostasis shared a similar expression pattern to BoFLC1, which was highly expressed at high temperature (27 °C). Of note, these genes were less expressed in HT than HS broccoli at 22 °C. Co-expression analysis identified a model for LRR-RLKs in survival-reproduction tradeoffs by modulating MAPK- versus phytohormones-signaling during head formation. The difference in head-forming ability in response to heat stress between HT and HS broccoli may result from their differential transcriptome profiles of LRR-RLK genes. High temperature induced JA- as well as suppressed auxin- and cytokinin-related pathways may facilitate a balancing act to ensure fitness at 27 °C. BoFLC1 was less expressed in HT than HS at 22 °C, whereas other FLC homologues were not. Promoter analysis of BoFLC1 showed fewer AT dinucleotide repeats in HT broccoli. These results provide insight into the early activation of stress- or development-related pathways during head formation in broccoli. The identification of the BoFLC1 DNA biomarker may facilitate breeding of HT broccoli. CONCLUSIONS: In this study, HT and HS broccoli genotypes were used to determine the effect of temperature on head formation by transcriptome profiling. On the basis of the expression pattern of high temperature-associated signaling genes, the HS transcriptome may be involved in stress defense instead of transition to the reproductive phase in response to heat stress. Transcriptome profiling of HT and HS broccoli helps in understanding the molecular mechanisms underlying head-forming capacity and in promoting functional marker-assisted breeding.