MYB transcription factors are one of the largest transcription factor families in higher plants, and they are involved in the regulation of a variety of plant life activities. Recent studies have shown that MYB transcription factors play a regulatory role in various stages of flower development and different parts of flower organs. In order to study the role of MYB transcription factor involved in ear development in wheat (Triticum aestivum L.), we isolated the TaMYB35 gene by taking advantage of the latest wheat genome data and the sequence homolog with the Arabidopsis thaliana AtMYB35 gene, which allowed identifying three copies TaMYB35A, TaMYB35B and TaMYB35D. Sequence analysis showed that TaMYB35A, TaMYB35B and TaMYB35D contained complete open reading frames of 924bp, 927bp and 927bp, respectively, encoding for 307, 308 and 308 amino acids, respectively. All the three members contained two MYB domains at the N-terminus, which are the feature of the R2R3-MYB transcription factor in the MYB gene family. Phylogenetic analysis showed that TaMYB35A was grouped with the orthologue of Triticum dicoccum, independent of a phylogenetic cluster where TaMYB35B and TaMYB35D were resided. TaMYB35A, TaMYB35B and TaMYB35D were found with higher transcripts in stamens, less in spikelets (excluding stamens) and no detectable transcripts in other tissues. Moreover, a significant difference in the expression of TaMYB35A was observed in samples harvested at sterile and fertile environments. Thus, this study clarified transcriptional characteristics of TaMYB35 in the development of wheat anthers, which thus provided insight for deciphering the molecular basis in wheat breeding with two-line hybrid strategy.