In this study, the effects of different extraction temperatures and food systems on the traceability and identification of gelatin from grass carp scales were investigated. Based on the sequence of grass carp type I collagen, 268 and 56 common characteristic peptides were obtained at different extraction temperatures and food systems, respectively. Further, 75 common and 41 unique characteristic peptides unaffected by extraction temperature were identified in bulk freshwater fish gelatin and grass carp gelatin, respectively. Additionally, 17 common and 10 unique peptides unaffected by food systems were obtained. Among these peptides, 2 common characteristic peptides of bulk freshwater fish gelatin that were unaffected by extraction temperature and food systems, and 8 unique characteristic peptides of grass carp gelatin that were unaffected by extraction temperature and food systems were consistently detected. In addition, the combination of common and unique peptides could improve the accuracy of fish gelatin traceability and identification.