AbstractA long‐standing and fundamental question in biology is how genes influence complex phenotypes. Combining near‐isogenic line mapping with genome expression profiling offers a unique opportunity for exploring the functional relationship between genotype and phenotype and for generating candidate genes for future study. We used a whole‐genome microarray produced with ink‐jet technology to measure the relative expression level of over 21 500 genes from an Arabidopsis thaliana near‐isogenic line (NIL) and its recurrent parent. The NIL material contained two introgressions (bottom of chromosome II and top of chromosome III) of the Cvi‐1 ecotype in a Ler‐2 ecotype genome background. Each introgression ‘captures’ a Cvi allele of a physiological quantitative trait loci (QTL) that our previous studies have shown increases transpiration and reduces water‐use efficiency at the whole‐plant level. We used a mixed model anova framework for assessing sources of expression variability and for evaluating statistical significance in our array experiment. We discovered 25 differentially expressed genes in the introgression at a false‐discovery rate (FDR) cut‐off of 0.20 and identified new candidate genes for both QTL regions. Several differentially expressed genes were confirmed with QRT–PCR (quantitative reverse transcription–polymerase chain reaction) assays. In contrast, we found no statistically significant differentially expressed genes outside of the QTL introgressions after controlling for multiple tests. We discuss these results in the context of candidate genes, cloning QTL, and phenotypic evolution.