Standard protocols aimed at identifying subclones of interest from bacterial artificial chromosomes (BACs) include the use of hybridization methods that are time consuming and often require the use of radioactive isotopes. Through our efforts to identify microsatellites in BACs from rainbow trout (Oncorhynchus mykiss) we have developed a nonradioactive polymerase chain reaction (PCR)-based screening technique to select microsatellites containing subclones for marker development. Two BACs were subcloned and screened by PCR using a vector-specific primer and a mix of microsatellite repeat primers. The subclones were then sequenced to evaluate the efficiency of the PCR screening method. Correlation between positive PCR amplification and presence of microsatellites varied between the two BACs (21.9% and 71.4%), but still a sufficient number of subclones were identified to enable design and optimization of microsatellite markers.