The genus Deuterodon has been taxonomically recently redefined. Seven of its species have been included in this genus, whereas three others are regarded as Incertae Sedis in Characidae. One of latter is Deuterodon pedri, an endemic species from Santo Antônio River in Minas Gerais. Cytogenetic and molecular data on 21 specimens of D. pedri allowed to reevaluate the phylogenetic position of this taxon. The specimens showed karyotypic formula 12m+12sm+20st+6a, low heterochromatin content, nucleolar organizer regions present in two chromosome pairs, and 10 and 2 labeled regions by fluorescence in situ hybridization with 18S and 5S probes respectively, a karyotype similar to Deuterodon stigmaturus. The phylogenetic tree generated by a cytochrome oxidase subunit I fragment revealed that D. pedri and Deuterodon singularis are closely related and are the sister-group of Deuterodon parahybae, Deuterodon iguape, Astyanax giton, Astyanax intermedius, Astyanax ribeirae, Astyanax scabripinnis, and Probolodus heterostomus. The same arrangement was partially recovered with the nuclear gene RAG2. Our data indicate the genus Deuterodon is not monophyletic and that it is closely related to other species that occur in Brazil coastal basins.