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A chromosome-specific satellite DNA from the South American fish species Leporinus obtusidens has been isolated and characterized. Sequence analysis and Southern hybridization studies indicate that the cloned 483-bp fragment is 60% AT rich and appears to comprise two diverged monomers. A highly variable low-copy number polymorphism was detected and, thus, this satellite DNA may serve as a valuable genetic marker. Using a Southern blot approach, the cloned satellite DNA cross-hybridized strongly to the DNA of Leporinus elongatus but failed to detect homologous sequences in the genomes of other closely related Leporinus species and higher vertebrates. Using fluorescence in situ hybridization to mitotic metaphase spreads of L. obtusidens and L. elongatus, this satellite DNA was located to the (peri)centromeric region of one single chromosome pair in both species. As the cloned satellite DNA sequence clearly evolved along a chromosomal lineage and is highly variable, it may serve as a very useful marker in further genetic, molecular and cytogenetic studies of the genus Leporinus.