The root-knot (Meloidogyne spp.) and reniform (Rotylenchulus spp.) nematodes are sedentary root parasites of cotton that cause considerable annual yield losses. To date, there is limited availability of genetic resistance to root-knot nematode in commercial cotton varieties and none available for the reniform nematode. An in vitro hairy root experimental system for studying the interaction between these nematodes and their cotton host would provide a sterile environment from which biological material could be collected without the space, labor and risk of secondary infections associated with greenhouse and growth chamber studies. We present here methods for propagating cotton hairy roots and nematode inoculation that facilitate in vitro nematode infection and reproduction. The reniform nematode has proven to be particularly recalcitrant to in vitro infection studies, therefore, the method presented here should make this pathogen more amenable to molecular-based techniques that are commonly implemented in studying host-nematode interactions.