Quantitative trait loci (QTL) analyses for five groups of hormones, including cytokinins in Arabidopsis roots were performed using recombinant inbred lines (Ler×Cvi). Significant QTLs were detected for cytokinins, jasmonic acid and salicylic acid. Separate analysis of two sub-populations, viz., vegetative and flowering plants revealed that many of the QTLs were development-specific. Using near-isogenic lines, several significant QTLs were confirmed; three co-localized QTL regions were responsible for determining several cytokinin metabolites. Using a knock-out plant, a functional role of zeatin N-glucosyltransferase gene (UGT76C2) underlying a large-effect QTL for levels of tZ-N-glucosides and tZRMP was evaluated in the metabolism of cytokinins. Pleotropic effects of this gene were found for cytokinin levels in both roots and leaves, but significant changes of morphological traits were observed only in roots. Hormone QTL analysis reveals development-specific and organ-dependent aspects of the regulation of plant hormone content and metabolism.