Objective: To determine whether congenital cytomegalovirus (CMV) infection is an etiologic factor in the pathogenesis of enlarged vestibular aqueducts (EVA). Design: Two different cohort studies. Subjects: The study population comprised 19 subjects with a history of congenital CMV infection and sensorineural hearing loss (cohort 1); 39 subjects with nonsyndromic EVA and their unaffected mothers (cohort 2); and 16 control subjects with EVA associated with Pendred syndrome and bi-allelic mutations of the SLC26A4 gene and their unaffected mothers. Results: In cohort 1, we detected EVA in 0 of 19 subjects with congenital CMV infection and sensorineural hearing loss. In cohort 2, anti-CMV serologic profiles were consistent with possible congenital CMV infection in 10 (26%) of 39 subjects with nonsyndromic EVA and 6 (38%) of 16 control subjects with Pendred syndrome (P= .52). These seroprevalence rates are similar to those expected in the general population (40%). Conclusion: In spite of their auditory phenotypic similarities, congenital CMV infection is not a significant factor in the etiology of EVA.