Abstract The classic plant growth‐promoting phytohormone cytokinin has been identified and established as a mediator of pathogen resistance in different plant species. However, the resistance effect of structurally different cytokinins appears to vary and may regulate diverse mechanisms to establish resistance. Hence, we comparatively analysed the impact of six different adenine‐ and phenylurea‐type cytokinins on the well‐established pathosystem Nicotiana tabacum ‐ Pseudomonas syringae . The efficiency of resistance effects was evaluated based on impacts on the host plant defence response by scoring infection symptoms and the direct impact on the pathogen by assessment of proliferation in planta . To identify common and cytokinin‐specific components involved in resistance effects, transcriptome profiling and targeted metabolomics were conducted in leaves treated with the different cytokinins. We observed clearly different potentials of the tested cytokinins in either suppressing infection symptoms or pathogen proliferation. Gene regulation and metabolite analyses revealed cytokinin‐type specific impacts on defence components, such as salicylic acid and related signalling, expression of PR proteins, and regulation of specialised metabolism. Cytokinins also strongly affected plant cell physiological parameters, such as a remarkable decrease in amino acid pools. Hence, this study provides comparative information on the efficiency of diverse cytokinins in mediating resistance in one well‐studied pathosystem and insights into the specific regulation of resistance effects mediated by different cytokinin molecules. This is particularly relevant for studies on the function of cytokinins or other phytohormones and compounds interacting with cytokinin activities in the context of pathogen infections and other stress scenarios, considering the diverse cytokinins present in plants.