We applied nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) in a single Trionyx triunguis reference specimen to identify diagnostic COl1 peptide markers. Our analysis revealed four COL1 biomarkers that differentiate T. triunguis from all sea turtle species, as well as seven additional turtle species with COL1 sequences available on NCBI: Chelonoidis abingdonii, Chrysemys picta bellii, Gopherus evgoodei, Mauremys reevesii, Pelodiscus sinensis, Terrapene carolina triunguis, and Trachemys scripta elegans. These findings enhance biomolecular identification methods for freshwater and marine turtle remains in archaeological and ecological contexts (especially on the Levant).