Inhibitory glycine receptors (GlyRs) are composed of homologous α- (α1–4) and β-subunits. The β-subunits (GlyRβ) interact via their large cytosolic loops with the postsynaptic scaffolding protein gephyrin and are therefore considered essential for synaptic localization. In situ hybridization studies indicate a widespread distribution of GlyRβ transcripts throughout the mammalian central nervous system (CNS), whereas GlyRα mRNAs and proteins display more restricted expression patterns. Here we report the generation of a monoclonal antibody that specifically recognizes rodent GlyRβ (mAb-GlyRβ) and does not exhibit crossreactivity with any of the GlyRα1–4 subunits. Immunostaining with this antibody revealed high densities of punctate GlyRβ immunoreactivity at inhibitory synapses in mouse spinal cord, brainstem, midbrain, and olfactory bulb but not in the neocortex, cerebellum, or hippocampus. This contrasts the abundance of GlyRβ transcripts in all major regions of the rodent brain and suggests that GlyRβ protein levels are regulated posttranscriptionally. When mAb-GlyRβ was used in double-labeling experiments with GlyRα1-, α2-, α3-, or α4-specific antibodies to examine the colocalization of GlyRβ with these GlyR subunits in the mouse retina, >90% of the GlyRα1–3 clusters detected were found to be GlyRβ-immunoreactive. A subset (about 50%) of the GlyRα4 puncta in the inner plexiform layer, however, was found to lack GlyRβ and gephyrin immunostaining. These GlyRα4-only clusters were apposed to bassoon immunoreactivity and hence synaptically localized. Their existence points to a gephyrin-independent synaptic localization mechanism for a minor subset of GlyRs. J. Comp. Neurol. 520:3962–3981, 2012. © 2012 Wiley Periodicals, Inc.