<p>Gene expression and the content of hormones regulating feed intake and digestion are crucial for understanding gastric evacuation (GE) and feeding frequency in fish. This study assessed the effects of temperature and size on these parameters by examining the domestication of steelhead trout (<i>Oncorhynchus mykiss</i>) at four temperatures (8 °C, 12 °C, 16 °C, and 20 °C) and with two sizes (75.99 g±10.48 g and 140.21 g±22.08 g) over two weeks. The trout were fasted for 72 h before the GE trial to ensure complete gastric emptying. In each GE trial, the expressions of peptide tyrosine (<i>PYY</i>), cholecystokinin (<i>CCK</i>), and <i>ghrelin</i> genes, along with corresponding serum hormone levels, were measured before feeding and at 0 h and 24 h after feeding, in conjunction with feed intake and 24-h gastric content mass. Results revealed significant effects of temperature on the expression of the three gastric genes. <i>CCK</i> expression increased at 8 °C, correlating with reduced feed intake, while <i>PYY</i> and <i>CCK</i> increased at 20 °C, correlating with accelerated gastric evacuation. Size significantly influenced serum levels of all three hormones, but the interaction between temperature and size did not affect <i>PYY</i> and <i>ghrelin</i> expression or their serum contents. This study provides molecular insights into how temperature and size regulate the digestion of steelhead trout, offering a theoretical framework for optimizing feeding frequency in aquaculture practices.</p>