Thymosin β4 (Tβ4) is a low molecular weight peptide found in several mammalian tissues and is known mainly by its ability to bind cytoskeletal actin, influencing cell migration and differentiation, and promoting tissue repair. Considering the functional role of this peptide, the main goal of this work was to characterize Tβ4 in mammals' saliva by using evolutionary and proteomic tools. For this, mammalian Tβ4 sequences were retrieved from NCBI, SwissProt and Ensembl databases. The alignment of Tβ4 amino acid sequences showed a high degree of conservation between species. The gene seems to be evolving under negative selection as indicated by a dN/dS ratio of 0.05. Whole saliva was collected from dog, human, rabbit, cow, horse and sheep and the salivary peptides were isolated through filtration and analyzed by LC-MS/MS. Spectra was processed against the database constructed with the retrieved Tβ4 sequences. For the first time, the identification of this peptide was achieved in rat, dog, horse and bovine saliva. Detection in these mammal species and its amino acid conservation suggest an important role of Tβ4 in the homeostasis of the mammalian oral cavity.