Bacterial leaf spot and bacterial leaf blight are global threats to the cultivation of cruciferous vegetables, and it is necessary to develop methods to easily detect, identify, and distinguish the causative pathogens Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca). Here, we used the sequence specificity of the exchangeable effector loci flanking the hrp gene cluster to design primers that can help detect and discriminate between Psm and Pca. Primers common to both bacteria (hrpK_fw1 and hrpK_fw2) were designed within hrpK at the end of the hrp gene cluster. Psm-specific primers (MAC_rv1 and MAC_rv2) were designed in hopPtoB1 and Pca-specific primers (ALS_rv1 and ALS_rv2) were designed in hopX1 adjacent to hrpK. PCR using hrpK_fw1 and MAC_rv1 or hrpK_fw2 and MAC_rv2 amplified DNA fragments of only Psm, P. syringae pv. tomato (causal agent of tomato bacterial speck), and P. syringae pv. spinaciae (causal agent of spinach bacterial leaf spot), among 76 strains of phytopathogenic bacteria. PCR using hrpK_fw1 and ALS_rv1 or hrpK_2 and ALS_rv2 amplified DNA fragments of only Pca. Multiplex PCR with these primers could easily distinguish Psm and Pca from bacterial colonies isolated on growth media and detect the pathogen in symptomatic leaves. Multiplex nested PCR with the primers detected contamination in one Psm- and/or one Pca-infected seeds in 1000 seeds. These results suggest that these PCR primers could help detect and discriminate Psm and Pca.