Embryonic root (radicle) development in the mature embryo following germination is essential for the formation of the root organ in plants. In this study a phenotype described by a lack of proper radicle development was identified in an intraspecific hybrid of Populus tomentosa Carr.. Association of this trait with Amplified Fragment Length Polymorphisms (AFLPs) markers was investigated in a segregating F1 population generated by intraspecific-controlled crossing between a highly fertile female P. tomentosa clone “5082” and a male P. tomentosa clone “JY”. A total of 3193 seeds were obtained, and the rate of germination found to be 48.74% at 15 to 20 days. 376 (24%) of seedlings were shown to lack a root organ following visual assessment of the developing radicle. Genetic regulation of this trait appeared to be via a single dominant gene or a set of tightly linked genes, based on the 3:1 ratio of the rooting versus nonrooting seed embryos. A Bulked Segregant Analysis approach using 5600 AFLP markers was applied to this population and revealed 2 AFLP markers, EcoRI + GAG/ Mse I + AAT-492 and EcoRI + GAG/Mse I + CCA-502, that were associated with the radicle development-controlling locus in P. tomentosa. The AFLP markers identified have potential for application in hybrid breeding via marker assisted selection, and provide a starting point for map based cloning of the radical development-controlling gene.