Abstract: The objective of the study was to catalog hair shaft abnormalities in individuals with ectodermal dysplasia (ED) syndromes using light microscopy and to compare findings with those in unaffected controls. Light microscopy was performed in a nonblinded manner on hair shafts from 65 participants with seven types of ED (hypohidrotic ED, ED–ectrodactyly–cleft lip or palate, ankyloblepharon–ectodermal defects–cleft lip and palate, Clouston syndrome, Goltz syndrome, Schopf-Schulz Passarge syndrome, and oculodentodigital dysplasia) and 41 unaffected controls. Hair donations were collected at the 28th Annual National Family Conference held by the National Foundation for Ectodermal Dysplasia. Control participants were recruited from a private dermatology practice and an academic children’s hospital outpatient dermatology clinic. Sixty-five affected participants and 41 unaffected controls were included in the analysis. We assessed the hair shafts of ED and control participants for abnormalities visible using LM. Light microscopy identified various pathologic hair shaft abnormalities in each type of ED, although none of the findings were statistically significantly different from those of the control group. Light microscopy is a poor adjuvant tool in the diagnosis of ED syndromes. Most findings are nonspecific and not sufficiently sensitive.