Genetic modification of the rooting system may lead to more drought tolerant upland cotton.This study was conducted to identify and map quantitative trait loci (QTLs) conferring root architecture in an interspecific cross G. hirsutum x G. barbadense population. A genetic map covering 3190.50 cM, with 219 marker loci and 26 linkage groups, was constructed by using F2 population. The Two F2 populations were grown in tube and pots experiments. Tap root traits (length, weight, and lateral root numbers) were evaluated. QTL analysis via composite interval mapping detected 17 QTLs, for three root traits including 3 QTLs for fresh root weight, 6 QTLs for lateral root numbers, and 2 QTLs for tap root length in tube experiment, while in pot experiment, 3 QTLs for fresh root weight and 3 QTLs for lateral root numbers were identified. Common QTLs were indentified for fresh root weight and lateral root numbers on chr. 18 & chr. 25 respectively, in both pot and tube experiments. The QTLs for root traits, firstly detected in Gossypium, may provide a basis for marker-assisted selection to improve productivity in root-crop breeding.