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Abstract Background Nicotiana benthamiana is widely used as a model plant to study plant-pathogen interactions. Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), participate in mediating plant growth, development and response to abiotic stress. However, the members of FLAs in N. benthamiana and their response to plant pathogens are unknown. Results 38 NbFLAs were identified from a genome-wide study. NbFLAs could be divided into four subclasses, and their gene structure and motif composition were conserved in each subclass. NbFLAs may be regulated by cis-acting elements such as STRE and MBS, and may be the targets of transcription factors like C2H2. Quantitative real time polymerase chain reaction (RT-qPCR) results showed that selected NbFLAs were differentially expressed in different tissues. All of the selected NbFLAs were significantly downregulated following infection by turnip mosaic virus (TuMV) and most of them also by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000), suggesting possible roles in response to pathogenic infection. Conclusions This study systematically identified FLAs in N. benthamiana, and indicates their potential roles in response to biotic stress. The identification of NbFLAs will facilitate further studies of their role in plant immunity in N. benthamiana.