LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYL INOSITOL-ANCHORED PROTEIN 2 (LYM2) is a GPI-anchored LysM receptor-like protein (LysM-RLP) that harbors three extracellular LysM-domains and a GPI moiety at the C-terminus. LYM2 in Arabidopsis thaliana is involved in chitin-mediated plasmodesmal flux regulation. The poplar paralogs PcLYM2-1 and PcLYM2-2 were identified and characterized in this study. PcLYM2-2 exhibits tissue-specific alternative splicing resulting in variants that differ exclusively at the first exon encoding the three LysM domains. Chitin-binding assays showed that all identified PcLYM2 proteins bind chitin, but with different capacity. Subcellular localization studies in Nicotiana benthamiana indicated that all identified PcLYM2 proteins localize at plasmodesmata, suggesting a role of these proteins in plasmodesmata-related functions. Plasmodesmal flux analysis of wildtype poplar and Pclym2-1 Pclym2-2 double knockout lines showed that PcLYM2 proteins mediate chitin-triggered PD closure. Loss-of-function of PcLYM2-1 is sufficient to abolish chitin-induced PD closure, suggesting that PcLYM2-1 is the primary LysM-RLP in poplar involved in this process.