A set of 13 polymorphic microsatellites were developed using a next generation sequencing approach for the endangered Neotropical catfish Conorhynchus conirostris. Using only 1/16 of a shot-gun pyrosequencing reaction, we were able to obtain 3,796 sequences containing putative microsatellites motifs. Out of 20 selected loci for further optimization, 13 loci were successfully genotyped in 25 specimens from a locality in the São Francisco River, Southeast Brazil. All microsatellite loci were polymorphic, with 4-18 alleles per locus (mean = 10.5) and generally high values of heterozygosity (mean = 0.796). These polymorphic markers will be a valuable tool for captive breeding and stoking programs and for analyses of population connectivity and genetic structure in this endangered Neotropical catfish.