The small genome of Arabidopsis contains at least nine expressed β-tubulin (TUB) genes, in contrast to the large genomes of vertebrate animals, which contain a maximum of seven expressed β-tubulin genes. In this study, we report the structures of seven new TUB genes (TUB2, TUB3, TUB5, TUB6, TUB7, TUB8, and TUB9) of Arabidopsis. The sequences of TUB1 and TUB4 had been reported previously. Sequence similarities and unique structural features suggest that the nine TUB genes evolved by way of three branches in the plant β-tubulin gene evolutionary tree. Two genes (TUB2 and TUB3) encode the same β-tubulin isoform; thus, the nine genes predict eight different β-tubulins. In contrast to the α-tubulin (TUA) genes with their divergent intron patterns, all nine TUB genes contain 2 introns at conserved positions. Noncoding 3′ gene-specific hybridization probes have been constructed for all nine TUB genes and used in RNA gel blot analyses to demonstrate that all nine genes are transcribed. Two-dimensional protein immunoblot analyses have resolved at least seven different β-tubulin isoforms in Arabidopsis, indicating that most, if not all, of the TUB transcripts are translated.