The Amazon basin is the world’s largest hydrographic basin, in terms of both its total area and its species diversity, with more than 2,700 species of fish. Despite this diversity, the data available on the fish fauna of the Amazon basin are still relatively scant and incomplete, in particular from the streams and floodplain lakes of the lower Amazon, which may contain a large proportion of the still undescribed species of the basin. Many of these species are expected to be of interest to the ornamental fish market. The investigation of the diversity of potential ornamental fish using molecular tools is even more limited. Given this scenario, the present study employed DNA barcoding to investigate the diversity of ornamental fish found in two streams and a floodplain lake of the lower Amazon. The mitochondrially encoded cytochrome c oxidase I (MT-CO1) molecular marker was used to identify the taxa, in combination with morphological keys. A total of 51 ornamental species were identified, representing 13 families and three orders. A majority of the species were found at only one of the sampling points, which indicates that the distribution of the species is influenced by ecological factors. The most speciose order was the Characiformes, followed by the Cichliformes and Siluriformes, while the family with the greatest diversity of species was the Acestrorhamphidae (31.3% of the total number of species), followed by the Cichlidae (27.4%), and the Lebiasinidae (9.8%). One specie was registered in the region of the lower Amazon for the first time, and evidence was found of the possible existence of species not formally described of Aphyocharax, Astyanax, Apareiodon and Hemigrammus.