Neuroligins constitute a family of transmembrane proteins localized at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. They are involved in synaptic function and maturation and recent studies have linked mutations in specific human Neuroligins to mental retardation and autism. We isolated the human Neuroligin homologs in Danio rerio. Next, we studied their gene structures and we reconstructed the evolution of the Neuroligin genes across vertebrate phyla. Using reverse-transcriptase polymerase chain reaction, we analyzed the expression and alternative splicing pattern of each gene during zebrafish embryonic development and in different adult organs. By in situ hybridization, we analyzed the temporal and spatial expression pattern during embryonic development and larval stages and we found that zebrafish Neuroligins are expressed throughout the nervous system. Globally, our results indicate that, during evolution, specific subfunctionalization events occurred within paralogous members of this gene family in zebrafish. Developmental Dynamics 239:688–702, 2010. © 2009 Wiley-Liss, Inc.