Mutations in the Connexin 26 gene (GJB2/Cx26) are responsible for more than half of all cases of prelingual nonsyndromic recessive deafness in Caucasians. The carrier frequency of the 35delG-GJB2 mutation was found to be as high as 2-4% in the Mediterranean populations. Different GJB2 mutations were reported in the Moroccan patients with autosomal recessive nonsyndromic hearing loss; however, rare studies were carried out on the carrier frequencies of these mutations in the healthy populations. The aim of this study was to estimate the carrier frequencies of the GJB2 mutations in the Moroccan population. The molecular analysis of the 35delG mutation and other GJB2 sequence variations was performed in 386 healthy unrelated Moroccan individuals with no known hearing loss. Five GJB2 sequence variations at heterozygous state were found: two mutations, 35delG and 109G > A (V37I), and three polymorphisms, 79G > A (V27I), 341G > A (E114G), and 457G > A (V153I). The carrier frequency of the 35delG mutation was the highest with 2.07% [95% confidence interval (0.90-4.04%)], followed by that of the V37I mutation with 1.43% (0.06-5.39). The carrier frequency of V27I, E114G, and V153I changes was estimated to be 0.71% (0.01-4.34). This finding shows that the 35delG carrier frequency found here is similar to the one observed in Mediterranean populations. It provides new information about GJB2 carrier rates facilitating the diagnosis and the genetic counseling in the Moroccan population.