Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of lipid mediators with promising anti-diabetic and anti-inflammatory properties. Comprehensive screening and identification of FAHFAs in biological samples would be beneficial to the discovery of new FAHFAs and enable greater understanding of their biological functions. Here, we report the comprehensive screening of FAHFAs in rice and Arabidopsis thaliana by chemical isotope labeling-assisted liquid chromatography–mass spectrometry (CIL-LC–MS). Multiple reaction monitoring (MRM) was used for screening of FAHFAs. With the proposed method, we detected 49 potential FAHFA families, including 262 regioisomers, in tissues of rice and Arabidopsis thaliana, which greatly extends our knowledge of known FAHFAs. In addition, we proposed a strategy to identify FAHFA regioisomers based on their retention on a reversed-phase LC column. Using the proposed identification strategy, we identified 71 regioisomers from 11 FAHFA families based on commercial standards and characteristic chromatographic retention behaviors. The screening technique could allow for the discovery of new FAHFAs in biological samples. The new FAHFAs identified in this work will contribute to the in-depth study of the functions of FAHFAs.