Abstract Eukaryotic genomes contain large amounts of repetitive DNA sequences, such as tandemly repeated satellite DNAs (satDNAs). These sequences are highly dynamic and tend to be genus- or species-specific due to their particular evolutionary pathways, although there are few unusual cases of conserved satDNAs over long periods of time. Here, we used multiple approaches to reveal that a satDNA named CharSat01-52 originated in the last common ancestor of Characoidei fish, a superfamily within the Characiformes order, approximately 140–78 million years ago, while its nucleotide composition has remained considerably conserved in several taxa. We show that 14 distantly related species within Characoidei share the presence of this satellite DNA, which is highly amplified and clustered in subtelomeric regions in a single species (Characidium gomesi), while remained organized as small clusters in all the other species. Defying predictions of the molecular drive of satellite evolution, CharSat01-52 shows similar values of intra- and interspecific divergence. Although we did not provide evidence for a specific functional role of CharSat01-52, its transcriptional activity was demonstrated in different species. In addition, we identified short tandem arrays of CharSat01-52 embedded within SMRT long reads of Astyanax paranae (536 bp to 3.1 kb) and A. mexicanus (501 bp to 3.9 kb). Such arrays consisted of head-to-tail repeats and could be found interspersed with other sequences, inverted sequences or neighbored by other satellites. Our results provide a detailed characterization of an old and conserved satDNA, challenging general predictions of satellite DNA evolution.