Previous studies based on DNA sequences of mitochondrial (mt) rRNA genes showed three main groups within the subfamily Serrasalminae: (1) a "pacu" clade of herbivores (Colossoma, Mylossoma, Piaractus); (2) the "Myleus" clade (Myleus, Mylesinus, Tometes, Ossubtus); and (3) the "piranha" clade (Serrasalmus, Pygocentrus, Pygopristis, Pristobrycon, Catoprion, Metynnis). The genus Acnodon was placed as the sister taxon of clade (2+3). However, poor resolution within each clade was obtained due to low levels of variation among rRNA gene sequences. Complete sequences of the hypervariable mtDNA control region for a total of 45 taxa, and additional sequences of 12S and 16S rRNA from a total of 74 taxa representing all genera in the family are now presented to address intragroup relationships. Control region sequences of several serrasalmid species exhibit tandem repeats of short motifs (12 to 33 bp) in the 3' end of this region, accounting for substantial length variation. Bayesian inference and maximum parsimony analyses of these sequences identify the same groupings as before and provide further evidence to support the following observations: (a) Serrasalmus gouldingi and species of Pristobrycon (non-striolatus) form a monophyletic group that is the sister group to other species of Serrasalmus and Pygocentrus; (b) Catoprion, Pygopristis, and Pristobrycon striolatus form a well supported clade, sister to the group described above; (c) some taxa assigned to the genus Myloplus (M. asterias, M tiete, M ternetzi, and M rubripinnis) form a well supported group whereas other Myloplus species remain with uncertain affinities (d) Mylesinus, Tometes and Myleus setiger form a monophyletic group.