In the present work, the compositions of the 27 triacylglycerols (TAGs) as well as fatty acid composition of Chilopsis linearis seed oil were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with diode array and mass spectrometric detections. It was shown that the main conjugated acid in the seed oil is a catalpic acid, (9E,11E,13Z)-octadeca-9,11,13-trienoic acid, while two unusual fatty acids have the parameters satisfying the literature data for (10E,12E)-octadeca-10,12-dienoic and (9E,12E)-octadeca-9,12-dienoic acids. For the two conjugated acid substituents the isosbestic point (at 234 nm) was found and was used to calculate the ratios of TAGs with different chromophores in the oil, taking into account the peak areas on the oil chromatogram.