Synaptosome-associated protein of 25 kDa (Snap25) is an intracellular protein that is defined as a target receptor for synapse vesicles prior to neurotransmitter release. Snap25 is highly conserved, with 61% identity between human and Drosophila melanogaster. Whereas mammals and chicken have a single locus for Snap25, the tetraploid goldfish has at least three loci. We report that the zebrafish has two loci with 91% amino acid identity to each other. The alternative splicing of exon 5 arose before the gene duplication. The expression patterns of the two loci are virtually identical in adult zebrafish. The two zebrafish snap25 loci are located in paralogous linkage groups that seem to correspond to human chromosome 20, which harbors the SNAP locus, and human chromosome 14. Because no additional Snap25 homologue has been reported for any mammal or chicken, snap25.2 may have been lost in the amniote or even tetrapod lineage.