The highly conserved Groucho/Tle gene family has widespread functions during embryonic development and in adults. For mammalians, four full-length Tle paralogues are known, whereas the whole spectrum of this gene family in fish species has not been analysed yet. Most detailed data exist for medaka, where 3 Tle genes have been described, Tle1, Tle3, and Tle4. We now isolated 3 additional Tle genes from the medaka genome. Sequence analysis identifies these genes as Tle2a, Tle2b, and Tle3b. Database searches of genomic sequences revealed an identical set of Tle paralogues being present in distantly related fish species, indicating duplicated Tle2 and Tle3 genes for the complete teleost lineage. Like the previously analysed medaka Tle genes, the three new genes show a broad expression pattern during embryogenesis. Nevertheless, a detailed comparison of all six Tle genes reveals critical differences in certain aspects of their expression pattern. In particular, we concentrated on the activity of Tle genes during ear development and found Tle2a and Tle2b expressed in this sensory organ. Developmental Dynamics 234:143–150, 2005. © 2005 Wiley-Liss, Inc.