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Chromosome substitution strains (CSSs) have been proposed as a simple and powerful way to identify quantitative trait loci (QTLs) affecting developmental, physiological, and behavioral processes. Here, we report the construction of a complete CSS panel for a vertebrate species. The CSS panel consists of 22 mouse strains, each of which carries a single chromosome substituted from a donor strain (A/J) onto a common host background (C57BL/6J). A survey of 53 traits revealed evidence for 150 QTLs affecting serum levels of sterols and amino acids, diet-induced obesity, and anxiety. These results demonstrate that CSSs greatly facilitate the detection and identification of genes that control the wide diversity of naturally occurring phenotypic variation in the A/J and C57BL/6J inbred strains.