The chromosomes of the primitive South American teleost fish Hoplias malabaricus have been analyzed by classical cytogenetic (C-, AgNOR-, Hoechst 33258-, and Q-banding) techniques. A highly repetitive DNA family has been cloned and sequenced. It is a tandemly repeated sequence of about 355 bp, yielding an overall base pair composition of 67% AT with long runs of > 50% As and 70% Ts. Analysis of sequence variation has allowed the further categorization of Hoplias satellite DNA into two evolutionary related subfamilies A and B, distinguishable by characteristic insertions and deletions within this 355-bp monomer. Subfamily A satellite is found (in diverged form) at the centromeres of most H. malabaricus chromosomes. Sequence variants are clustered on specific chromosomal subsets. Subfamily B satellite is highly specific for the paracentromeric heterochromatin on one particular chromosome pair by fluorescence in situ hybridization. These results indicate that the Hoplias satellite DNA family has evolved in a concerted manner predominantly via recombination events involving homologous, rather than non-homologous chromosome regions. The clones isolated here may be useful for the molecular, genetic, and cytological analysis of the genus Hoplias.