The scattered distribution pattern of microsatellites is a challenging problem in fish cytogenetics. This type of array hinders the identification of useful patterns and the comparison between species, often resulting in over-limited interpretations that only label it as "scattered" or "widely distributed". However, several studies have shown that the distribution pattern of microsatellites is non-random. Thus, here we tested whether a scattered microsatellite could have distinct distribution patterns on homeologous chromosomes of closely related species. The clustered sites of 18S and 5S rDNA, U2 snRNA and H3/H4 histone genes were used as a guide to compare the (GATA)nmicrosatellite distribution pattern on the homeologous chromosomes of sixTrachelyopterusspecies:T.coriaceusandTrachelyopterusaff.galeatusfrom the Araguaia River basin; T.striatulus,T.galeatusandT.porosusfrom the Amazonas River basin; andTrachelyopterusaff.coriaceusfrom the Paraguay River basin. Most species had similar patterns of the (GATA)nmicrosatellite in the histone genes and 5S rDNA carriers. However, we have found a chromosomal polymorphism of the (GATA)nsequence in the 18S rDNA carriers ofTrachelyopterus galeatus, which is in Hard-Weinberg equilibrium and possibly originated through amplification events; and a chromosome polymorphism inTrachelyopterusaff.galeatus, which combined with an inversion polymorphism of the U2 snRNA in the same chromosome pair resulted in six possible cytotypes, which are in Hardy-Weinberg disequilibrium. Therefore, comparing the distribution pattern on homeologous chromosomes across the species, using gene clusters as a guide to identify it, seems to be an effective way to further the analysis of scattered microsatellites in fish cytogenetics.