In previous studies, we employed multiple behavior assays, including propensity to feed, simulated trawl capture and escape response, to prove the presence of bold and shy personality in olive flounder. However, the molecular mechanism of the different personality has not been elucidated. In the present study, the transcriptomic profiles of the hindbrain from flounder with distinct personalities were compared. A total of 144 differently expressed genes were identified, including 74 up-regulated and 70 downregulated genes. Genes involved in hypoxia stress were detected in SP flounder, accompanied by down-regulation of ribosomal RNA synthesis. In addition, genes related to calcium signaling pathway, including endothelin, b-Fos, c-Fos and c-Jun were up-regulated in SP flounder. Furthermore, personality-related genes, including UI, CCK, c-Fos showed a significantly higher level in SP flounder compared with BP flounder. GO enrichment analysis indicated that the GO categories “the tight junction pathway” and “lipid transport or localization pathway” are enriched in SP flounder, suggesting that the central nervous system homeostasis would be compromised. Finally, a simple and scalable DNA methylation profiling allows for methylation analysis for different genes. The results found that part of gene expression is negatively related to methylation of promoter. Altogether, identification of the related genes in flounder with different personalities will shed new light to improve critical industry issues related to stress and increase aquaculture production of flounder. Overall design: Olive flounder (101.06 ± 6.39 g, mean ± SD) were obtained in central experimental station of Chinese academy of fisheries sciences (Hebei, China), and preserved in aquarium facilities in aerated 500 L tanks under a 12:12 light: dark cycle at 18 °C at Shanghai Ocean University. Fish were fed to satiation twice daily (8:00 and 18:00) with commercial fish pellets. Bold personality (BP) and shy personality (SP) flounder were selected according to methods previous reported (Rupia et al, 2016b). Fish were humanly killed by anesthesia with 0.1% 2-phenoxyethanol, and the blood was collected from the caudal 114 vein with the heparinized syringess. The blood was diluted and the red blood cell (RBC) was count on a microscope. The concentration of haemoglobin in blood was measured using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Hindbrain tissues were collected and stored in liquid nitrogen for further analysis. Total RNA was extracted from each sample using Trizol Reagent according to the manufacturer’s protocol, and was treated with DNaseI to eliminate potential genomic DNA contamination. The concentration and integrity were were determined by spectrophotometry (NanoDrop ND-1000 system, Thermo Fisher Scientific, DE, USA)) and gel electrophoresis. Equal amounts of RNA from the hindbrain were obtained from six individuals. The Illumina TruSeq mRNA Stranded Sample Preparation kit was used in constructing the Illumina sequencing libraries according to previous methods (Zhang et al., 2017). The libraries were then sequenced on Illumina HiSeq 2000 using a 100-bp paired-end sequencing module.