Tarantula venom predominantly comprises peptides and small proteins (venom toxins). To explore this further, we generated a combined gland cDNA library from five different tarantula species. Subsequently we randomly sequenced 752 clones, which led to the identification of sixteen distinct toxin groups, including one novel group. Each group consists of a minimum of three identical sequences. A comprehensive analysis of these toxin groups was carried out, and we simulated the 3D structures of certain predicted toxins. These transcripts can be used to the production of venom toxins through recombinant techniques, offering promising applications in various biomedical applications.