Despite the broad use of single-cell/nucleus RNA sequencing in plant research, accurate cluster annotation in less-studied plant species remains a major challenge due to the lack of validated marker genes. Here, we generated a single-cell RNA sequencing atlas of soil-grown wheat roots and annotated cluster identities by transferring annotations from publicly available datasets in wheat, rice, maize, and Arabidopsis. The predictions from our orthology-based annotation approach were next validated using untargeted spatial transcriptomics. These results allowed us to predict evolutionarily conserved tissue-specific markers and generate cell type-specific gene regulatory networks for root tissues of wheat and the other species used in our analysis. In summary, we generated a single-cell and spatial transcriptomics resource for wheat root apical meristems, including numerous known and uncharacterized cell type-specific marker genes and developmental regulators. These data and analyses will facilitate future cell type annotation in non-model plant species.