Gene family identification from ESTs can be a valuable resource for analysis of genome evolution but presents unique challenges in organisms for which the entire genome is not yet sequenced. We have developed a novel gene family identification method based on negative selection patterns (NSP) between family members to screen EST-generated contigs. This strategy was tested on five known gene families in Arabidopsis to see if individual paralogs could be identified with accuracy from EST data alone when compared to the actual gene sequences in this fully sequenced genome.