The systematic development of functional molecular markers requires a set of non-redundant ESTs. However large-scale EST sequencing projects are frequently based on random sequencing of shotgun cDNA clones, which usually results in high redundancy of ESTs. To increase the efficiency in the identification of novel ESTs/genes for the development of non-redundant markers, it is necessary to reduce the redundancy of sequencing by eliminating the redundant clones from the cDNA library. In order to reach this goal, we adopted the 'total cDNA hybridization' approach on a set of barley cDNAs from different libraries to remove redundant clones prior to sequencing. To quantify the accuracy of the approach we performed cluster analysis on the ESTs of the clones used in this study. The results show that up to 90% of the redundant clones that are present in clusters can be identified by this approach. Furthermore cluster analysis revealed that the set of non-hybridizing cDNA clones contains 82-89% 'singletons'. Therefore, cDNA hybridization of arrayed colony filters represents an efficient and cheap procedure to deplete a cDNA library of redundant clones in order to generate non-redundant molecular markers. In addition, this approach is suitable for maximizing the rate of gene discovery in an EST programme.