The one-lined pencilfish Nannostomus unifasciatus is a small fish from the flooded forests of the Amazon basin. Pencilfish are popular in aquaria and are used as ornamental fishery resource by riverine communities from central Amazonia. Here we report a set of nine microsatellite loci for N. unifasciatus. Number of alleles and heterozygosity per locus in a sample of 30 pencilfish ranged from 4 to 19 and from 0.39 to 0.86, respectively. These markers will prove useful to investigate population genetic structure, identify conservation units, and to conduct phylogeographical reconstructions in pencilfish from the flooded forests of Amazonia.