Rice is an important crop in the word, and fat is one of the main important nutrient components of rice. The lipid content and fatty acid composition of grains significantly influences the quality of rice. In this study, 94 homozygous recombination inbred lines (RILs) were developed and the crude fat content of them displayed a normal distribution ranging from 0.44% to 2.62%. Based on their taste quality, a positive association between fat content and eating quality was revealed. Then, two lines (FH and FL) were selected with similar agronomic characteristics and different lipid content and taste quality for RNA sequencing analysis, and a total of 619 differentiable expressed genes were detected, primarily enriched in metabolic pathways such as starch and sucrose metabolism, fatty acid metabolism, and amino acid metabolism. The expression of two genes related to fatty acid synthesis and elongation was significantly up-regulated, while the expression of three genes related to fatty acid degradation was significantly down-regulated in FH grains. By using liquid chromatography, the relative levels of palmitic acid and oleic acid were discovered significantly higher in FH grains. Additionally, the comparative genomic analysis was conducted to visualize genomic differences of five genes. Ultimately, two genes (Os07g0417200 and Os12g0102100) were selected to be the key gene to affect the lipid metabolism, especially for the synthesis of unsaturated fatty acids, significantly changing the eating quality of rice. These results provide a theoretical basis for improving the taste quality of rice.