Abstract: In order to study the role of PfNLR gene family in Paulownia fortunei resistance to Paulownia witches' broom(PaWB), bioinformatics methods were used to comprehensively analyze the composition, physicochemical properties, chromosomal localization, evolution, structure, cis-acting elements and tissue expression specificity of P. fortunei NLR (PfNLR) family members. Combined with the miRNA and transcriptome data before and after phytoplasma infecting Paulownia, the genes associated with resistance to PaWB were screened. The results showed that 199 PfNLRs in P. fortunei belonged to 6 subfamilies; 91.9% of PfNLRs were located on 18 chromosomes of P. fortunei, of which 62.8% were clustered, among them, there were 10 chromosome segment repeats and 1 chromosome tandem repeats in 23 PfNLRs; PfNLR contains hormonal and stress elements; integrated transcriptome and miRNA analysis found that there were 156 pairs of targeted relationships between PfNLRs and miRNAs; most of PfNLRs were expressed in buds and roots; of which, 16 PfNLRs were significantly different before and after phytoplasma infection. Further analysis revealed that PfNLR181 mediated by pf-miRNA482 played a role in resistance of PaWB. These results could provide a theoretical basis for analyzing the function of PfNLR protein.