We describe a method for efficient parallel mapping of expressed sequence tag (EST) sites onto yeast artificial chromosome (YAC) clones. The strategy involves an initial YAC clone pooling scheme that minimizes the number of required PCR amplifications. This is followed by parallel analysis of PCR amplicons of EST sequences. Using this method, we have screened 600 EST sites in combinatorial pools of 3449 YAC clones that contain Arabidopsis thaliana DNA inserts. The presence of these genes on YACs was detected by amplifying EST sequences with PCR and analyzing the reaction products by agarose gel electrophoresis. Of the 600 ESTs, 271 were found to map to individual YACs. Software tools are presented that allow for the automated analysis of this electrophoresis data. Suggestions for the scale-up of this method to map large genomes are discussed.