Tomosyn was previously identified as a syntaxin‐binding protein that inhibits soluble NSF (n‐ethylmaleimide‐sensitive fusion protein) attachment protein receptor (SNARE)‐mediated secretion. We set out to investigate the distribution of tomosyn mRNA in the mammalian brain and found evidence for the presence of two paralogous genes designated tomosyn‐1 and ‐2. In a collection of tomosyn‐2 cDNA clones, we observed four splice variants (named xb‐, b‐, m‐ and s‐tomosyn‐2) derived from the skipping of exons 19 and 21. This feature is conserved with tomosyn‐1 that encodes three splice variants. To compare the expression pattern of tomosyn‐1 and ‐2, we performed in situ hybridization experiments with gene‐specific probes. Both genes were expressed in the nervous system, clearly following distinct spatial and developmental expression patterns. Real‐time quantitative PCR experiments indicated that tomosyn‐1 expression was up‐regulated less than threefold between developmental stages E10 and P12, whereas tomosyn‐2 expression increased 31‐fold. Not only the transcription level, but also the splice composition of tomosyn‐2 mRNA shifted during development. We conclude that two distinct genes drive expression of seven tomosyn isoforms. Their expression patterns support a role in regulating neuronal secretion. All isoforms share conserved WD40 and SNARE domains separated by a hypervariable module, the function of which remains to be clarified.