Summary Phosphorylation of receptor kinase (RK) is pivotal for signaling in pattern-triggered immunity (PTI). The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2) is a conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22), initiating plant defense pathways. However, the dynamic FLS2 phosphorylation regulation at the plasma membrane in response to flg22 needs further elucidation. Through single-particle tracking, we demonstrated that the Ser-938 phosphorylation site influences flg22-induced FLS2 spatiotemporal dynamics and dwell time. Förster resonance energy transfer-fluorescence lifetime (FRET-FLIM) imaging microscopy, coupled with protein proximity indexes (PPI), revealed increased co-localization of FLS2/FLS2S938D-GFP with AtRem1.3-mCherry in response to flg22. In contrast, FLS2S938A-GFP shows no significant changes, indicating that Ser-938 phosphorylation influences the efficient FLS2 sorting into AtRem1.3-associated microdomains. Significantly, Ser-938 phosphorylation enhanced flg22-induced internalization and immune responses, thus demonstrating its regulatory role in FLS2 partitioning into functional AtRem1.3-associated microdomains for activating flg22-induced plant immunity.