Background: Chromosomal rearrangements are thought to be an important driving force underlying lineage diversification, but their link to speciation continues to be debated. Antarctic teleost fish of the family Nototheniidae (Notothenioidei) diversified in a changing environmental context, which led to ecological, morphological, and genetic differentiation among populations. In addition, extensive chromosomal repatterning accompanied species divergence in several clades. The most striking karyotypic changes involved the recent species radiation (about 10 My) of the genus Trematomus, with chromosomal pair numbers ranging between 29 and 12. These dramatic reductions in chromosome number resulted mostly from large-scale chromosome fusions. Multiple centric and/or tandem fusions have been hypothesized in at least seven of the twelve recognized Trematomus species. To reconstruct their evolutionary history, we employed comparative cytogenomics (BAC-FISH and chromosome painting) to reveal patterns of interspecific chromosomal orthologies across several notothenioid clades.Results: We defined orthologous chromosomal segments of reference, termed Structural Units (SUs). SUs were identified in a total of 18 notothenioid species. We demonstrated for the first time that SUs were strongly conserved across every specimen examined, with chromosomal syntenies highlighting a paucity of intrachromosomal macro-rearrangements. Multiple independent fusions of these SUs were inferred in the Trematomus species, in contrast to the shared SU fusions in species of the sister lineage Notothenia.Conclusions: The SU segments were defined units of chromosomal rearrangement in the entire family Nototheiidae, which diverged from the other notothenioid families 20 My ago. Some of the identified chromosomal syntenies within the SUs were even conserved in their closest relatives, the family Eleginopsidae. Comparing the timing of acquisition of the fusions in the closely related genera Notothenia and Trematomus of the nototheniid species family, we conclude that they exhibit distinct chromosomal evolutionary histories, which may be relevant to different speciation scenarios.