The objective of this investigation was to determine the prevalence of Cx26 mutations in familial and sporadic cases of non-syndromic preverbal hearing impairment (HI). Molecular analysis of the Connexin 26 (Cx26/GJB2) gene was performed in 271 non-consanguineous individuals from the north of Italy, enrolled in the study because of the presence of non-syndromic preverbal sensorineural HI. One hundred and forty-six subjects (group 1) were referred from different ENT, paediatric and clinical genetic services, while 125 individuals (group 2) underwent Cx26 analysis based on precise anamnestic and clinical criteria for non-syndromic HI and low risk of acquired deficit. All of the cases were also classified as familial or sporadic due to the presence or absence of other documented childhood HI in the family. Of the total 271 individuals, 36.9% were positive for Cx26 mutations: 37 belonged to group 1 and 63 to group 2, which delineates a statistically significant difference between the two groups. The difference is mainly attributable to sporadically occurring cases. No significant differences between group 1 and group 2 were found regarding the prevalence of the common 35delG variant and the number of unidentified putative Cx26 alleles, although these latter were shown to be higher in sporadically occurring cases of the unselected group 1. The difference observed in Cx26 prevalence can be explained by the clinical selection of group 2, which ensures minimum risk of including cases of acquired HI. In particular, in cases of sporadically occurring HI, the use of a defined protocol increases the chances of a positive molecular result, improving genetic counselling and the possibility of establishing better genotype-phenotype correlation. Our data raise questions about the possible interpretation of Cx26 heterozygosity in a selected population of hearing-impaired individuals.