These experiments used custom-made Agilent oligonucleotide (55- to 60-mer) microarrays representing 18,850 unique consensus GLEAN gene predictions from the D. pseudoobscura genome. For each species/subspecies, RNA from 7-day-old virgin males and females of 3-4 pooled isofemale lines was used (D. pseudoobscura, MV2-25, Flagstaff 18, Mather 10, and Mather 32; D. persimilis, MSH 3, Mather G, and MSH 42; and D. pseudoobscura bogotana, Toro 7, Susa 6, and Potosi 3). Three replicate hybridizations were performed for each (sub)species and sex. Hybridization was performed using single-color labeling. Statistical analysis was performed using ANOVA. dN and dS values between D. pseudoobscura and D. persimilis were calculated with PAML and provided by the authors. Raw microarray data are available from the GEO database under series accession number GSE17192.