Guard cells (GCs) regulate gas exchange and water loss in plants and have been extensively studied in Arabidopsis thaliana. However, cross-species comparisons at single-cell resolution remain limited. To address this, we aimed to define conserved and divergent transcriptomic signatures of GCs by generating a comparative single-cell atlas encompassing five species: A. thaliana, soybean (Glycine max), tomato (Solanum lycopersicum), rice (Oryza sativa), and sorghum (Sorghum bicolor). We performed single-nucleus RNA sequencing (sNucRNA-seq) on leaf tissues from each species, followed by integrative bioinformatic analyses to identify and annotate GC populations. Orthology-informed transcriptomic comparisons and co-expression analyses were applied to assess shared and species-specific regulatory modules. Our analyses revealed a conserved core transcriptome in GCs across species, enriched for genes involved in stomatal movement, abscisic acid signaling, carbon dioxide signaling, reactive oxygen species metabolism, and ion transport. Conserved expression of transcription factors (e.g. MYB60, FAMA orthologs), transporters (e.g. KAT1, SLAC1), and stress-responsive genes was observed, despite considerable evolutionary divergence. These findings provide a reference framework for cross-species GC biology, reveal conserved regulatory features, and nominate candidate genes for functional validation. This work contributes molecular insights toward improving drought resilience and stomatal efficiency in crop species through targeted gene engineering.