The ionotropic glutamate receptor (iGluR) gene family has been widely studied in animals and is determined to be important in excitatory neurotransmission and other neuronal processes. We have previously identified ionotropic glutamate receptor-like genes (GLRs) in Arabidopsis thaliana, an organism that lacks a nervous system. Upon the completion of the Arabidopsis genome sequencing project, a large family of GLR genes has been uncovered. A preliminary phylogenetic analysis divides the AtGLR gene family into three clades and is used as the basis for the recently established nomenclature for the AtGLR gene family. We performed a phylogenetic analysis with extensive annotations of the iGluR gene family, which includes all 20 Arabidopsis GLR genes, the entire iGluR family from rat (except NR3), and two prokaryotic iGluRs, Synechocystis GluR0 and Anabaena GluR. Our analysis supports the division of the AtGLR gene family into three clades and identifies potential functionally important amino acid residues that are conserved in both prokaryotic and eukaryotic iGluRs as well as those that are only conserved in AtGLRs. To begin to investigate whether the three AtGLR clades represent different functional classes, we performed the first comprehensive mRNA expression analysis of the entire AtGLR gene family. On the basis of RT-PCR, all AtGLRs are expressed genes. The three AtGLR clades do not show distinct clade-specific organ expression patterns. All 20 AtGLR genes are expressed in the root. Among them, five of the nine clade-II genes are root-specific in 8-week-old Arabidopsis plants.