Myostatin is an extremely potent negative regulator of vertebrate skeletal muscle development. A phylogenetic analysis suggests that salmonids should possess four distinct genes, although only MSTN-1 orthologs have been characterized. Described herein are the rainbow trout (rt) MSTN-2a and -2b genes and subsequence analysis of their promoters and their quantitative expression profiles. Both genes are similarly organized, contain several putative myogenic response elements, and are legitimate MSTN-2 orthologs based on Bayesian analyses. However, rtMSTN-2b contains two in-frame stop codons within the first exon and unspliced variants of both transcripts were expressed in a tissue-specific manner. Complete splicing of rtMSTN-2a occurred only in brain, where expression is highest, whereas rtMSTN-2b transcripts were mostly present in unspliced forms. The presence of stop codons in the rtMSTN-2b open reading frame and the expression of mostly unspliced transcripts indicate that this particular homolog is a pseudogene. These results confirm our previous phylogenetic analysis and suggest that all salmonids likely possess four distinct myostatin genes. The tissue-specific expression and differential processing of both rtMSTN-2 transcripts as well the pseudogenization of rtMSTN-2b may reflect compensatory and adaptive responses to tetraploidization and may help limit rtMSTN-2a's influences primarily to neural tissue.