Thymic mimetic cells are molecular hybrids between medullary-thymic-epithelial cells (mTECs) and diverse peripheral cell types. They are involved in eliminating autoreactive T cells and can perform supplementary functions reflective of their peripheral-cell counterparts. Current knowledge about mimetic cells derives largely from mouse models. To provide the high resolution that proved revelatory for mice, we performed single-cell RNA sequencing on purified mimetic-cell compartments from human pediatric donors. The single-cell profiles of individual donors were surprisingly similar, with diversification of neuroendocrine subtypes and expansion of the muscle subtype relative to mice. Informatic and imaging studies on the muscle-mTEC population highlighted a maturation trajectory suggestive of skeletal-muscle differentiation, some striated structures, and occasional cellular groupings reminiscent of neuromuscular junctions. We also profiled thymic mimetic cells from zebrafish. Integration of data from the three species identified species-specific adaptations but substantial interspecies conservation, highlighting the evolutionarily ancient nature of mimetic mTECs. Our findings provide a landscape view of human mimetic cells, with anticipated relevance in autoimmunity. Copyright © 2024 Elsevier Inc. All rights reserved.