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Comparative chromosome-painting analysis among highly rearranged karyotypes of Sigmodontinae rodents (Rodentia, Cricetidae) detects conserved syntenic blocks, which are proposed as chromosomal signatures and can be used as phylogenetic markers. In the Akodontini tribe, the molecular topology (Cytb and/or IRBP) shows five low-supported clades (divisions: “<i>Akodon</i>”, “<i>Bibimys</i>”, “<i>Blarinomys</i>”, “<i>Oxymycterus</i>”, and “<i>Scapteromys</i>”) within two high-supported major clades (clade A: “<i>Akodon</i>”, “<i>Bibimys</i>”, and “<i>Oxymycterus</i>”; clade B: “<i>Blarinomys</i>” and “<i>Scapteromys</i>”). Here, we examine the chromosomal signatures of the Akodontini tribe by using <i>Hylaeamys</i> <i>megacephalus</i> (HME) probes to study the karyotypes of <i>Oxymycterus amazonicus</i> (2n = 54, FN = 64) and <i>Blarinomys breviceps</i> (2n = 28, FN = 50), and compare these data with those from other taxa investigated using the same set of probes. We strategically employ the chromosomal signatures to elucidate phylogenetic relationships among the Akodontini. When we follow the evolution of chromosomal signature states, we find that the cytogenetic data corroborate the current molecular relationships in clade A nodes. We discuss the distinct events that caused karyotypic variability in the <i>Oxymycterus</i> and <i>Blarinomys</i> genera. In addition, we propose that <i>Blarinomys</i> may constitute a species complex, and that the taxonomy should be revised to better delimit the geographical boundaries and their taxonomic status.