Abstract: Gelatins were digested with trypsin. Mass spectrometric analysis indicated that the molecular weight range of the gelatins shifted from 40,000 Da to less than 5,000 Da. The peptides in the digest mixtures were identified with high performance liquid chromatography-electrospray ionization mass spectrometry. It was found that the digest mixtures of bovine contained lots of typical peptides, which may act as markers for gelatin differentiation. The bovine gelatin was identified with marker peptides that corresponding bovine collagen type I. The applicability of the method was validated with mixed gelatins. The result indicated that tryptic digestion and identification of marker peptides with mass spectrometry is a feasible strategy for gelatin differentiation.