The β subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone β subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHβ subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHβ subunit was particularly high throughout the radiation of mammals while FSH and TSHβ subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHβ subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.