BACKGROUND: Congenital cytomegalovirus (CMV) infection is the leading cause of non-genetic congenital hearing loss. The contribution of congenital CMV to prelingual deafness and the pathophysiology is largely unknown. OBJECTIVE: (1) To analyze the prevalence of congenital CMV among cochlear implant (CI) recipients with prelingual deafness. (2) To genotype CMV present in dried blood spots (DBS) and in the inner ear years after birth. STUDY DESIGN: Children and adults with prelingual deafness who received a CI in 2010-2011 were included prospectively. Perilymphatic fluids were collected during CI surgery and, in the pediatric cases, DBS were retrieved for CMV DNA detection. Furthermore, a cohort of children with prelingual deafness who received a CI between 2003 and 2008 were included retrospectively. CMV detection in DBS and perilymph was followed by gB and gH genotyping. RESULTS: Seventysix pediatric CI recipients were included. Seventy DBS were tested for CMV DNA, resulting in a prevalence of congenital CMV of 14% (10/70). Perilymphatic fluid was available from 29 pediatric CI recipients. One perilymph fluid, of a 21-month old girl with congenital CMV, asymptomatic at birth, was CMV DNA positive. The CMV strain in the perilymph was genotypically identical to the strain present in her DBS (gB1/gH2). Perilymph samples from 21 adult CI recipients were CMV DNA negative. CONCLUSIONS: Our study stresses the important contribution of congenital CMV among pediatric CI recipients. Furthermore, our genotyping data support the hypothesis that CMV-related hearing loss is associated with ongoing viral replication in the inner ear up to years after birth.