To determine the origin and evolutionary significance of a recently discovered isoform of the estrogen receptor (ER beta), we examined the phylogenetic relationship of ER beta to the well-known alpha isoform (ER alpha) and other steroid receptors. Our phylogenetic analyses traced the origin of ER beta to a single duplication event at least 450 million years ago. Since this duplication, the evolution of both ER isoforms has apparently been constrained such that 80% of the amino acid positions in the DNA binding domain (DBD) and 53% of the ligand binding domain (LBD) have remained unchanged. Using the phylogenetic tree, we determined the amount of evolutionary change that had occurred in two ER isoforms. The DBD and the LED had lower rates of evolutionary change compared to the NH2 terminal domain. However, even with strong selective constraints on the DBD and LED, our phylogenetic analyses demonstrate two clearly separate phylogenetic histories for ER alpha and ER beta dating back several hundred million years. The ancient duplication of ER and the parallel evolution of the two EP, isoforms suggest that, although ER alpha and ER beta share a substantial degree of sequence identity, they play unique roles in vertebrate physiology and reproduction.