Abstract This study describes the karyotypes of three species of the genus Peckoltia (Loricariidae: Ancistrini). Fishes were collected in the Jari (Peckoltia sp. 1 and Peckoltia sp. 2) and Xingu rivers (Peckoltia vittata (Steindachner, 1881)) in the Amazon rainforest. Karyotypes were 2n = 52 for Peckoltia vittata (FN=102: 16 metacentrics (m), 20 submetacentrics (sm), 14 subtelocentrics (st), 2 acrocentrics (a)) and Peckoltia sp. 2 (FN=102: 32 m + sm, 18st, 2a). Peckoltia sp. 1 (FN=102: 44 m + sm, 6st, 2a, 1B) had 2n = 53 due to a B chromosome. The species differ in chromosomal morphology. Nucleolar Organizer Regions (NOR) was identified within a distal region of the long arm of pair 9 in Peckoltia vittata, in pair 10 and in a homologue of pair 25 in Peckoltia sp. 1, as well as in pair 17 and in a homologue of pair 18 in Peckoltia sp. 2. Chromomycin A3 banding agreed with the location of the NORs. C-banding patterns revealed large non-centromeric heterochromatic blocks, probably of a common origin. Our results suggest a higher level of similarity between Peckoltia vittata and Peckoltia sp. 1.