The recognition of pathogens by the innate immune system relies on a wide range of inhibitory and activating receptors. Some of these non-rearranging receptors belong to the immunoglobulin superfamily (IgSF) and in teleost fish the novel immune-type receptor (NITR) and the novel immunoglobulin-like transcript (NILT) have been reported. Here we describe the identification and characterisation of three new NILTs from rainbow trout (Oncorhynchus mykiss), with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain. The cytoplasmic regions contain either immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or an immunoreceptor tyrosine-based activating motif (ITAM) for downstream signalling. Alignment of the various NILT Ig domains revealed a high similarity, especially between Ig domains from NILTs found in this study. Furthermore, a phylogenetic tree showed that NILTs are more closely related to the triggering receptor expressed on myeloid (TREM) cells and NKp44 than to NITRs. The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues. Expression of NILTs was also shown to be an early event in development, with both eyed eggs and embryos expressing all four genes. The results obtained in this study and future experiments will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in rainbow trout.