The glucose-regulated protein, 94 kDa (GRP94), is an endoplasmic reticulum (ER)-localized heat shock protein that plays among other functions a crucial role in folding and exports of Toll-like receptors (TLRs) and some other immune-relevant factors. We identified two copies of the GRP94-encoding gene in rainbow trout sharing 91% DNA sequence identity. The conceptually translated ORFs encode a 795-aa GRP94a and a 510-aa GRP94b protein variant, respectively, with characteristic domains and amino acid residues. However, the shorter variant lacks motifs required for its localization in the ER and might thus represent an isoform of the putative mammalian ortholog GRP94a. Heat stress only slightly affects the expression of the two GRP94-encoding trout genes, as reported for mammals. We recorded the abundances of transcripts coding for both GRP94 variants as well as for a broad panel of TLRs representing their potential targets. In embryonic and larval trout, only the mRNAs encoding TLR1, -2, -9, and -20 were found in significant concentrations, while the expression of nine other TLRs was hardly detectable. The GRP94a-encoding gene showed constantly high expression levels indicating that this isoform is vitally required throughout the life cycle of rainbow trout. The concentration of the GRP94b-encoding mRNA was only ~0.1% compared to the GRP94a mRNA level. These structural and gene expression data together suggest that the two GRP94 gene products fulfill different physiological roles.