Root proteins have not been examined extensively for cultivated and wild Gossypium species. This study identified unique cotton root proteins from G. hirsutum, G. barbadense, G. arboreum, and G. longicalyx in 2-D gels, which were then characterized by Q-TOF tandem mass-spectral sequencing. The subsequent in silico bioinformatics annotation of Q-TOF sequenced fragments from selected major spots revealed proteins that were associated with primary and secondary metabolism, defense and stress conditions, and growth and development functions. Most annotated proteins were common and shared among the species, but a few were unique to a species. The major constituents of root proteome included pathogenesis-related and latex proteins along with ubiquitously expressed proteins, such as 3-phosphoshikimate 1-carboxyvinyltransferase, NDP-kinase, and protease. Latex proteins, auxin-responsive proteins, actin, and annexin, which were annotated in this study, participate in cotton root elongation, growth, and regeneration processes. The differences in sequenced proteins of various cotton species were observed by spot intensity, concentration, and isoelectric points. This study was undertaken to document expressed proteins observed in the root that should be useful as a preliminary inventory of root proteome in different cotton species.