In order to resist pathogen infection, plants have established a set of autoimmune mechanisms during their evolution, including systemic acquired resistance (SAR) and induced systemic resistance (ISR) [18]. SAR is often induced by harmful factors, while ISR is induced by beneficial bacteria in soil [19; 20]. When pathogens attack plants and cause trauma, the plants induce SAR to ensure plants develop broad-spectrum disease resistance. ISR also has broad-spectrum resistance, depending on the interaction between plants and pathogens. Additionally, some enzymes play crucial roles in the recognition and response to plant pathogens, such as peroxididases (PRXs), mitogen-activated protein kinase (MPK), and callose synthase. These enzymes take part in the regulation pathways of signal transduction, cell wall formation, synthesis of reactive oxygen species (ROS), and hormone response [21; 22; 23]. MPK4 participates in the signaling pathway of SA and JA, and conversely, in _mpk4_ mutants, the signal transduction of JA is turned off, causing severe black spot disease in _Arabidopsis_[23]. _PMR4_ encodes glucan-type callose synthase, responsible for producing callose to prevent the invasion of pathogenic bacteria [24]. PRX33 and PRX34 negatively regulate the resistance to _A. brassicola_ in _Arabidopsis_, and thus the level of ROS and the plaques have been found to be decreased in _prx33_ and _prx34 Arabidopsis_ mutants [25]. Germplasm resources contain rich genetic variations. It is effective to improve breeding abilities by using elite germplasm [26]. Many excellent alleles cloned from natural genetic populations have been widely used in modern breeding of rice, maize, and other major staple crops. The _UPA2_ allele in the wild ancestor of maize can reduce leaf angle, with previous reporting showed that modern hybrids (Nongda 108) with the wild _UPA2_ allele enhance high-density maize yields [27]. _Waxy (Wx)_ gene encoding granule-bound starch synthase controls amylose synthesis in endosperm, and several _Wx_ alleles have been utilized in modern rice quality improvement [28]. However, it is relatively weak in broccoli breeding programs. Black spot disease is a common disease during the growth period of broccoli, but current research on black spot disease is still limited. In order to explore excellent genetic materials for improving black spot resistance, it is also required to carry out phenotypic identification of vegetable germplasm resources. Additionally, black spot disease and black rot disease in broccoli curd are phenotypically indistinguishable in the field, because they both display black lesions. Therefore, it is necessary to develop sensitive molecular markers to identify _A. alternata_. To this end, we first developed a molecular marker for detecting _A. alternata_ which infects broccoli. Then, we applied two techniques to identify the resistance to black spot disease in broccoli and screened out the optimal evaluation method. Additionally, we established the grade standards for the identification of black spot resistance in broccoli and evaluated the resistance to black spot disease in 173 broccoli germplasm resources. Furthermore, three high-resistance varieties and 55 resistance varieties were selected via phenotypic scoring, and these lines can be used to improve resistance to black spot disease in broccoli.