Studying gene expression in wheat–rye translocation lines is complicated due to the presence of homeologs in hexaploid wheat and high levels of synteny between wheat and rye genomes (Naranjo and Fernandez-Rueda, 1991 [1]; Devos et al., 1995 [2]; Lee et al., 2010 [3]; Lee et al., 2013 [4]). To overcome limitations of current gene expression studies on wheat–rye translocation lines and identify genome-specific transcripts, we developed a custom Roche NimbleGen Gene Expression microarray that contains probes derived from the sequence of hexaploid wheat, diploid rye and diploid progenitors of hexaploid wheat genome (Lee et al., 2014). Using the array developed, we identified genome-specific transcripts in a wheat–rye translocation line (Lee et al., 2014). Expression data are deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE58678. Here we report the details of the methods used in the array workflow and data analysis.