The objective of this study was molecular characterization of a set of deletion stocks and other aneuploids for use in chromosome bin mapping of ESTs in wheat. Wheat aneuploid stocks including 21 nullisomic-tetrasomic (NT), 24 ditelosomic (Dt), and 101 deletion (del) lines were screened with 526 EST clones. A total of 1,951 loci were detected by 493 informative EST clones and tagged 150 of the 159 deletion intervals or chromosome bins. Previously described deletion lines del1AS-4, del6AL-2, del6BS-6, and del7DS-6 were found to have normal chromosome constitution. The short arm deletion in del3AS-3 may be translocated from an unknown chromosome as this stock is nullisomic for the 3AS arm. Thirty-five new deletions were detected in 26 lines. Most of the new deletions occurred in terminal regions of chromosomes and probably resulted from the loss of very small terminal fragments that were difficult to detect cytologically. Eleven chromosome aberrations were also detected in two NT and five Dt lines. Overall, the chromosome bin map provides a resolution of around 28 Mb for an anchor map of a basic set of seven chromosomes of the Triticeae. Any target gene can be allocated to a specific 28-Mb bin and associated ESTs, anchored to the other Triticeae/grass maps including rice and, therefore, amenable to molecular cloning by comparative and wheat-based positional cloning methods.