Nonalcoholic fatty liver disease (NAFLD), the most prevalent chronic liver disease, is characterized by substantial variations in case-level severity. In this study, we used a genetically diverse Collaborative Cross (CC) mouse population model to analyze the global transcriptome and clarify the molecular mechanisms involved in hepatic fat accumulation that determine the level and severity of NAFLD. Twenty-four strains of male CC mice were maintained on a high fat/high sucrose (HF/HS) diet for 12 weeks and their hepatic gene expression profiles were determined by next-generation RNA-sequencing. We found that the development of the nonalcoholic fatty liver (NAFL) phenotype in CC mice coincided with significant changes in the expression of hepatic genes at the population level, evidenced by the presence of 724 differentially expressed genes involved in lipid and carbohydrate metabolism, cell morphology, vitamin and mineral metabolism, energy production, and DNA replication, recombination, and repair. Importantly, expression of 68 of these genes strongly correlated with the extent of hepatic lipid accumulation in the overall population of HF/HS diet-fed male CC mice. Results of Partial Least Squares (PLS) modeling showed that these derived hepatic gene expression signatures help to identify the individual mouse strains that are highly susceptible to the development of NAFLD induced by a HF/HS diet. These findings imply that gene expression profiling, combined with a PLS modeling approach, may be a useful tool to predict NAFLD severity in genetically diverse patient populations.