A total of 34 apple (Malus × domestica Borkh.) cDNA libraries were constructed from root, leaf, bud, shoot, flower, and fruit tissues, at various developmental stages and/or under biotic or abiotic stress conditions, and of several genotypes. From these libraries, 190,425 clones were partially sequenced from the 5? end and 42,619 clones were sequenced from the 3' end, and a total of 182,241 high-quality expressed sequence tags (ESTs) were obtained. These coalesced into 23,442 tentative contigs and 9843 singletons, for a total of 33,825 apple unigenes. Functional annotation of this unigene set revealed an even distribution of apple sequences among the three main gene ontology categories. Of ~33,000 apple unigenes, 8437 (25%) had no detectable homologs (E >0.1) in the Arabidopsis genome. When the entire apple unigene set was compared with the entire citrus [Citrus sinensis (L.) Osbeck] unigene set and the poplar (Populus trichocarpa Torr. & Gray) predicted proteome, both members of the core eudicot and rosids clade, 13,521 of apple unigenes matched one or more sequences in citrus, while 25,817 had counterparts in the poplar protein database. Apple–Arabidopsis–citrus–poplar comparisons revealed closer evolutionary relationships between apple and poplar than with the other two species. Genes involved in basic metabolic pathways appear to be largely conserved among apple, citrus, poplar, and Arabidopsis.