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<i>Ancistrus</i> is a highly diverse neotropical fish genus that exhibits extensive chromosomal variability, encompassing karyotypic morphology, diploid chromosome number (2n = 34–54), and the evolution of various types of sex chromosome systems. Robertsonian rearrangements related to unstable chromosomal sites are here described. Here, the karyotypes of two <i>Ancistrus</i> species were comparatively analyzed using classical cytogenetic techniques, in addition to isolation, cloning, sequencing, molecular characterization, and fluorescence in situ hybridization of repetitive sequences (i.e., 18S and 5S rDNA; U1, U2, and U5 snDNA; and telomere sequences). The species analyzed here have different karyotypes: <i>Ancistrus</i> sp. 1 (2n = 38, XX/XY) and <i>Ancistrus cirrhosus</i> (2n = 34, no heteromorphic sex chromosomes). Comparative mapping showed different organizations for the analyzed repetitive sequences: 18S and U1 sequences occurred in a single site in all populations of the analyzed species, while 5S and U2 sequences could occur in single or multiple sites. A sequencing analysis confirmed the identities of the U1, U2, and U5 snDNA sequences. Additionally, a syntenic condition for U2-U5 snDNA was found in <i>Ancistrus</i>. In a comparative analysis, the sequences of rDNA and U snDNA showed inter- and intraspecific chromosomal diversification. The occurrence of Robertsonian rearrangements and other dispersal mechanisms of repetitive sequences are discussed.