The invention belongs to the technical field of cotton disease resistance breeding and specifically relates to a cotton verticillium wilt resistant seedling stage identification method. The method provided by the invention comprises the following main steps of: firstly carrying out shaking culture on cotton verticillium wilt germ 6SY2-37 collected in China Center for Type Culture Collection with the collection number being CCTCC NO: M2011095 in a Czapek's liquid medium at the temperature of 25 DEG C for 5d, filtering by the use of gauze, counting by the use of a blood cell counting panel, preparing 5*106 spore/ml, pulling up seedling with a matrix when the water-planting cotton seedling grows to a stage of two leaves and one core, cutting 1cm of water root, dipping the root for 10s and inoculating, embedding wet soil into a plastic earthen bowl, inoculating pathogen, immediately watering to prevent the cotton seedling from fading, inoculating for 10 days and causing the disease. The method provided by the invention has advantages of high survival rate of the cotton seedling after inoculation, uniform inoculated pathogen, rapid morbidity, convenient operation and good stability of results. In addition, the method takes short time, requires short seedling growing time and is convenient to manage. Therefore, the method is suitable for cotton verticillium wilt resistance identification and pathogen pathogenicity differentiation determination of large-batch materials.