Despite compelling evidence that adaptation to local climate is common in plant populations, little is known about the evolutionary genetics of traits that contribute to climatic adaptation. A screen of natural accessions of Arabidopsis thaliana revealed Tsu-1 and Kas-1 to be opposite extremes for water-use efficiency and climate at collection sites for these accessions differs greatly. To provide a tool to understand the genetic basis of this putative adaptation, Kas-1 and Tsu-1 were reciprocally crossed to create a new mapping population. Analysis of F₃ families showed segregating variation in both δ¹³C and transpiration rate, and as expected these traits had a negative genetic correlation (r_g=− 0.3). 346 RILs, 148 with Kas-1 cytoplasm and 198 with Tsu-1 cytoplasm, were advanced to the F₉ and genotyped using 48 microsatellites and 55 SNPs for a total of 103 markers. This mapping population was used for QTL analysis of δ¹³C using F₉ RIL means. Analysis of this reciprocal cross showed a large effect of cytoplasmic background, as well as two QTL for δ¹³C. The Kas-1 × Tsu-1 mapping population provides a powerful new resource for mapping QTL underlying natural variation and for dissecting the genetic basis of water-use efficiency differences.