Cis-regulatory elements (CREs) are critical in coordinating the regulation of gene expression. Understanding how CREs evolve represents a challenging endeavor. Here, we constructed a comprehensive single-cell atlas of CRE activity in Oryza sativa, integrating data from 104,029 nuclei representing 128 discrete cell states across nine distinct organs. We used a comparative genomic approach to analyze cell-type-specific CRE activity between Oryza sativa and four additional grass species (Zea mays, Sorghum bicolor, Panicum miliaceum, and Urochloa fusca). We revealed a complex interplay whereby accessible chromatin regions (ACRs) had different levels of conservation dependent on if these regions were broadly accessible, or cell-type specific. We found that on average epidermal ACRs were less conserved in regions of synteny compared to other cell types, potentially indicating that large amounts of regulatory evolution have taken place in the epidermal layers of these species. We found a significant overlap between cell-type-specific ACRs and conserved non-coding sequences. Finally, we identified a subset of ACRs that were overlapping the repressive histone modification H3k27me3, which were conserved across evolutionary time, implicating them as potentially critical silencer CREs. Collectively, these results highlight the dynamic evolution of cell-type-specific CRE activity using comparative genomics. Read more