Temperature is a major environmental factor that influences growth, development, metabolism, and physiological performance in fish. Grass carp (Ctenopharyngodon idella) is a highly productive fish in freshwater culture. To understand the molecular mechanism of grass carp under heat stress, we used RNA-Seq to analyze the liver and brain transcriptome of 12 libraries constructed from high-temperature (36 °C) and control (28 °C) groups. We obtained 42.49 and 42.57 GB of clean data from six liver and six brain libraries, respectively, and identified 2,534 genes that were differentially expressed in liver tissue and 1622 in brain tissue (P < 0.05). According to KEGG analysis, significant differences occurred in the expression of genes involved in metabolic and immune pathways, such as the cAMP signaling pathway, apoptosis, calcium signaling pathway, lipid metabolism, and protein processing in endoplasmic reticulum and peroxisome proliferator-activated receptor signaling pathways. This study revealed that high temperature enhanced lipid metabolism, reduced fatty acid synthesis, and disrupted the immune system of grass carp. These results investigated the molecular regulation of heat stress in grass carp and provided valuable information for the healthy culture of grass carp under high temperatures.