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Chromosome formulae are listed for 433 Neotropical freshwater fish species from primary and secondary divisions (sensu Myers, 1938), including representatives from 145 genera in 33 families. Of the species analyzed, 11.3% present more than one chromosome number, 5.8% have morphologically differentiated sex chromosomes and 2.3% have supernumerary chromosomes. Comparison of the modal chromosome numbers of the subgroups of the Superorder Ostariophysi shows an evolutionary tendency towards higher chromosome numbers.