An analysis of transcriptional variation in the liver using a panel of B.A chromosome substitution strains identified 4209 transcripts that are differentially expressed relative to the C57BL/6J background and 1010 transcripts that are differentially expressed between C57BL/6J and A/J strains. A subset of these strains (substituting Chromosomes 1, 6, and 15) was used to identify 386 additional differentially expressed transcripts in the kidney. Approximately 15% of differentially expressed transcripts are located on the substituted chromosome. These cis-QTL are codirectionally expressed with the donor strain A/J. By comparison, trans-regulated loci comprise 85% of differentially expressed transcripts, often show opposite direction of change compared with A/J, and can be regulated by multiple chromosome substitutions. Gene expression differences in this study provide evidence for transgressive segregation: Only 438 of 4209 QTL in liver were inside the parental range. By combining QTL data with known biological functions, we were able to identify physiologic pathways altered in multiple strains. In many cases the same pathways were altered by multiple distinct chromosome substitutions. Taken together, these results suggest that widespread epistatic background effects may result in complex and overlapping transcriptional relationships among different chromosome substitution strains. Transcriptional profiling of chromosome substitution strains reveals a complex genetic architecture of transcriptional regulation.