Since most of the DEFLs were recently discovered and many are not well represented on standard microarrays, little is known about their expression pattterns. To address this gap, we have designed a custom Affymetrix microarray (AtMtDEFL array) with known and predicted Arabidopsis and Medicago DEFL genes and the closely related maternally expressed gene family. The custom array was utilized for hybridizations of mRNAs from a wide range of conditions and tissues from both plant species, enabling a comparative analysis in the two model plant species. The custom chip transcript analysis provided the first expression evidence for many of the computationally identified DEFLs previously lacking gene expression evidence, and indicated both common and unique patterns of DEFL gene expression between the two model plant species. The custom designed Affymetrix chip consisting of nearly all DEFLs from Arabidopsis thaliana was used for transcript profiling study in a variety of plant organs, developmental stages, and pathogenic associations totalling 16 different treatments. Each treatment was supported by three biological replicates giving a grand total of 48 samples.