The inhibitory ligand-gated ion channel family of receptors, including the type A γ-aminobutryic acid (GABAA) and glycine receptors, mediates inhibitory neurotransmissions in the central nervous system. In this study, GABA receptor (GABR) evolution was explored through comparative genomics using genomes that span divergent lineages. GABAA/Gly receptor–like (GRL) gene sequences were retrieved from the genomes of various species ranging from mammal to fish to worm and subjected to cross-species comparison. All vertebrate GRL gene sets in the study but no invertebrate ones exhibit the extensive and conserved pattern of gene clustering that is characteristic of human GABR genes, indicating that the gene clusters were established early in vertebrate evolution, after divergence from the invertebrates. Moreover, the vertebrate gene structure is highly conserved with a basic 9–coding exon structure, whereas, as well as being diverse in copy numbers and chromosomal loci, the invertebrate GRL genes display a variety of gene structures. Remarkably, the invertebrates each possess a unique GRL gene pair that lies in neighboring loci within their respective genomes: zc482.5 and zc482.1 in roundworm, CG8916 and CG17336 in fruitfly, Ci4249 and Ci4254 in Ciona, and these were revealed by phylogenetic analysis to be homologous to human GABR α and β subunits, respectively. The phylogenetic classification of these genes is also corroborated by experimental ligand-binding measurements using recombinant gene products. Furthermore, the 3 invertebrate gene pairs harbor characteristic key residues and exhibit similarities in intron positions to their vertebrate counterparts. The results strongly indicate that such a gene pair originally existed in the bilaterian ancestor from which all 3 phyla evolved and suggest that the extant GABR clusters arose from an ancestral α–β subunit gene pair gave rise to the extant GABR clusters.