The potential of microsatellite sequences as genetic markers in channel catfish (Ictalurus punctatus) was investigated with respect to their variability, inheritance, and usefulness in related species. Six small insert genomic DNA libraries enriched for six families of microsatellites of channel catfish were constructed. We describe here the isolation, characterization, and PCR amplification of 32 microsatellites from channel catfish. The flanking primer regions of microsatellite loci were highly conserved between channel catfish and blue catfish (I. furcatus). Of the 32 loci, 29 were amplified from blue catfish using primers designed from channel catfish, indicating conservation of primer binding sequences. Most of the amplified alleles from channel catfish and blue catfish were polymorphic. White catfish (Ameiurus catus) and flathead catfish (Pylodictus olivaris) loci were also amplified. The microsatellite markers are highly polymorphic for all catfish species tested and are inherited as codominant markers. They should be highly useful for construction of genetic linkage maps of catfish and for marker-assisted selection.