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Objective: To immunohistochemically characterize a group of oral myofibroblastic lesions (MLs) and to evaluate the ultrastructural features of myofibroblasts., Material and Methods: Using a tissue microarray technique (TMA), cases of myofibroma (MF), of nodular fasciitis (NF), of desmoplastic fibroma (DF), and of myofibroblastic sarcoma (MS) from the Universidad Autónoma Metropolitana Xochimilco, and a Private Oral Pathology Service in Mexico City were stained with antibodies against alpha-smooth muscle actin (α-SMA), H-caldesmon, vimentin, desmin, β-catenin, CD34, anaplastic lymphoma protein kinase (ALK-1), and Ki-67., Results: Nineteen of the 22 MF cases, 2/5 of the NF cases, 1/10 of the DF cases, and 1/2 of the MS cases were positive for α-SMA. 1/2 of the MS cases were positive for desmin; 6/10 of the DF cases were positive for β-catenin, and 2 of the MF cases were positive for ALK-1. All of the MLs were positive for vimentin and negative for H-caldesmon and CD-34. The Ki-67 labeling index in all of the 8/22 MF, 3/5 NF, and 2/2 MS cases was ≥10%. For all of the MLs evaluated, ultrastructural analysis revealed spindle-shaped cells containing endoplasmic reticulum and peripheral actin filament bundles., Conclusion: In certain myofibroblastic lesions, the use of auxiliary techniques (such as immunohistochemistry) can be critical for differential diagnosis., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)