Rainbow trout (Oncorhynchus mykiss) are an important commercial fish species in the United States, with a total value of fish sales received by U.S. trout growers in 2023 totaling $27.2 million. Understanding the mechanisms of growth regulation of trout can lead to improved commercial production advances. One proposed strategy is to alter one-carbon metabolism through dietary interventions that alter epigenetic pathways regulating growth performance. We recently demonstrated that maternal dietary choline intake positively affects offspring growth performance. This project focused on evaluating the effects of maternal dietary choline intake on global DNA methylation profiles and related transcriptional changes in rainbow trout offspring. Three experimental diets were formulated to test different levels of choline intake: (a) 2065 ppm choline (Low Choline, 0% supplementation), (b) 5657 ppm choline (Medium Choline, 0.6% supplementation), and (c) 9248 ppm choline (High Choline, 1.2% choline supplementation). Six rainbow trout families were fed experimental diets beginning 18 months post-hatch until spawning; their offspring were fed a commercial diet. Reduced representation bisulfite sequencing (RRBS) was utilized to measure genome-wide methylation in offspring immediately after hatching. When comparing to the Medium Choline offspring, differential DNA methylation occurred more in the Low Choline offspring than High Choline, especially in genic features like promoters. The differentially methylated CpGs (q ≤ 0.01) were identified evenly between CpG islands and shores in the genome, mostly found in the introns of genes. Genes such as fabp2 and leap2B associated with protein binding, fatty acid binding, DNA binding, and response to bacteria were differentially methylated and detected as differentially regulated genes by previous RNA-seq analysis. Although these findings indicate that levels of dietary choline available in broodstock diets alter offspring DNA methylation, most differentially expressed genes were not associated with differential DNA methylation, suggesting additional mechanisms playing a role in regulating gene expression in response to maternal choline intake.