Abstract Body: Atherosclerosis is a complex disorder characterized by plaque buildup in the arterial walls, which leads to constriction and reduced blood flow to vital organs. Identifying and characterizing the diverse cell types driving atherosclerosis is crucial for developing targeted therapies. Fibroblasts significantly impact atherosclerosis by regulating inflammation and maintaining the structural integrity of the plaque. However, identifying fibroblasts is challenging because other cell types within the lesion, such as vascular smooth muscle cells (VSMCs) and endothelial cells, can adopt fibroblast-like characteristics. Thus, there is a need for specific cell surface markers to identify and further characterize fibroblasts in atherosclerosis. To overcome this challenge, we used cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) in an atherogenic-prone mouse model with smooth muscle cell lineage tracing (ROSA26 ZsGreen1^+/+; Ldlr^−/−; Myh11-CreERT2), allowing us to distinguish fibroblasts from fibroblast-like cells derived from VSMCs. We identified one fibroblast-like population derived from VSMCs and three fibroblast populations, including a subset characterized by specific expression of CD26. The CD26⁺ fibroblast subpopulation exhibited unique migratory properties, enabling its movement from the adventitia to the lesion during the development of atherosclerosis. Previous studies have identified CD26⁺ in dermal fibrosis, indicating that this fibroblast lineage plays a role in wound healing in various disease contexts. Further studies will be necessary to determine the role of CD26⁺ fibroblasts in atherogenesis. In conclusion, by employing high-dimensional cell surface phenotyping of fibroblasts in atherosclerosis, we identified CD26 as a highly expressed and specific marker of a migratory fibroblast subpopulation in atherosclerosis progression.