Thermo-photoperiod-sensitive genic male sterile (TPGMS) wheat is important in utilization of heterosis. To facilitate the use of such wheat line in agriculture, more knowledge about molecular mechanisms of TPGMS genes is required. In this study, we set up a normalized complementary DNA (cDNA) library based on the strategy of saturation hybridization with genomic DNA using TPGMS wheat line. This normalized cDNA library consists of cDNA from six directionally cloned cDNA libraries constructed with spike and anther tissues from spike developmental stages. From the normalized cDNA library, 3,264 single-pass expressed sequence tag (EST) were obtained. Exclusion of sequences shorter than 100 bp resulted in 3,223 vector-trimmed ESTs with a mean length of 926 bp. Clustering and assembly analysis resulted in 2,175 unique ESTs from 423 contigs and 1,752 singletons. Taking advantage of various tools and database, gene function classification showed that 60% of the ESTs were predicted to have putative gene function. Of the 2,175 unique ESTs, 264 (12%) displayed significant homology (BlastX E values <10-5) to genes previously reported to be involved in cold-response related processes. Among these, sequences encoding activities related to primary metabolism, signal transduction, and transcriptional regulation were observed. Finally, in the total EST sequences, 108 potential SSRs were found. The unigene dataset will now be used to fabricate biochips carrying all identified genes for TPGMS wheat functional genomic research.