Understanding the patterns and processes driving natural genetic variation in gene expression is of fundamental importance to biology. In this study, we examined genetic variation in gene transcription through expression QTL (eQTL) analysis in the Tsu-1 x Kas-1 recombinant inbred line (RIL) mapping population of Arabidopsis thaliana. To understand how natural variation in transcription responds to abiotic stress, we conducted eQTL in both well watered and soil drying conditions. Further, we evaluated whether elements of genome structure were associated with eQTL occurance and genes responding to treatment conditions. Overall, we identified thousands of genes that responded to soil moisture availability and hundreds of eQTLs. However, we identified very few interactions between eQTLs and environmental conditions, and both treatment conditions were enriched for similar gene ontology (GO) categories. We did find strong evidence for associations between genome structure and natural variation in transcription. In general, genes with eQTLs were positively associated with local recombination rates and levels of polymorphism while genes responding to the treatment were negatively correlated with these factors. Our study provides further insight into the origin and maintenance of natural variation in transcription and how that variation responds to environmental conditions. Expression analysis by hybridization to atSNPTILE array (Affymetrix). Seed of the 104 RILs from reciprocal crosses between A. thaliana (L.) Heynh. accessions Kas-1 (CS903) and Tsu-1 (CS1640) were sown on fritted clay (Profile Products LLC, Buffalo Grove, IL) in 2.5-inch pots. Plants were arranged in a randomized complete block design consisting of 4 blocks, and then the pots were refrigerated at 4°C in darkness for 6 d to cold-stratify the seeds prior to commencement of a 12 h photoperiod in Conviron ATC60 growth chambers (Controlled Environments, Winnipeg, MB), at 23°C and 40% humidity during the day and 20°C and 50% humidity during the dark period. Light intensity was approximately 330 µmol m-2 s-1. After four weeks of growth, half of the plants were given a drought treatment, while the others remained fully watered. The drought treatment was randomly assigned to 2 of the 4 blocks and consisted of a slow decrease in soil moisture content over the course of one week. Each day, all pots assigned to the drought treatment were weighed, and water was added to individual pots to bring them to the target gravimetric water content. The target water content decreased each day, in the following series: 100%, 90%, 80%, 70%, 60%, 45%, and 40% of field capacity. At the end of the treatment, leaf tissue was collected from both treatments for RNA extraction.