To identify the main spoilage bacterium on fresh-cut leafy vegetables and establish a multiplex PCR assay. Based on physiological-biochemical, molecular identification, and artificial contamination tests, the main bacterium to spoil fresh-cut leafy vegetables was identified as Exiguobacterium spp. and Exiguobacterium acetylicum. Comparative genomics showed that P401_RS0117025 and oxi_50,582,462 genes are specific to Exiguobacterium spp. and E. acetylicum. Based on this, three pairs of primer sets to EaG-291, EaS-2B, and Ea16S-12 genes were designed and used to develop a multiplex PCR assay, which exhibited 100% specificity among 16 Exiguobacterium and 10 non- Exiguobacterium strains. Finally, 84 fresh-cut leafy vegetable samples were analyzed by multiplex PCR assay and standard physiological-biochemical experiments, the results showed multiplex PCR assay reached a detection rate of 96%. The main spoilage bacterium was identified as Exiguobacterium spp. and E. acetylicum on fresh-cut leafy vegetables based on the novel specific genes explored in this study. A rapid, specific, and sensitive PCR assay was developed for the detection of Exiguobacterium spp. and E. acetylicum. • The first study to identify the main spoilage bacteria on fresh-cut leafy vegetables are Exiguobacterium spp. and E. acetylicum. • Discovery of specific detection genes of Exiguobacterium spp. and E. acetylicum. • A triple PCR method for detecting Exiguobacterium spp. and E. acetylicum. • Established method has good prospects in the quality detection of FCLVs. [ABSTRACT FROM AUTHOR]