DFNB1 deafness, caused by mutations in the gene encoding connexin-26 (GJB2), is the most frequent subtype of autosomal recessive non-syndromic hearing impairment. Molecular testing for GJB2 mutations has become a standard diagnostic approach for subjects with this disorder. However, 10-50% of affected subjects with GJB2 mutations carry only one mutant allele. A 309 kb deletion truncating the GJB6 gene (encoding connexin-30) was shown to be the accompanying mutation in up to 50% of deaf GJB2 heterozygotes in different populations. We report the molecular characterisation of the breakpoint junction of a novel 232 kb deletion in the DFNB1 locus, del(GJB6-D13S1854), which was also found in trans with pathogenic GJB2 mutations in affected subjects. The deletion arose by unequal homologous recombination, involving an AluY sequence inside GJB6 intron 2, a mechanism which might generate other deletions at DFNB1. We developed a novel diagnostic test for the combined detection of del(GJB6-D13S1830) and this new del(GJB6-D13S1854) in a single PCR assay. The del(GJB6-D13S1854) mutation accounts for 25.5% of the affected GJB2 heterozygotes which remained unresolved after screening for del(GJB6-D13S1830) in Spain, 22.2% in the UK, 6.3% in Brazil and 1.9% in northern Italy. It was not found in affected GJB2 heterozygotes from France, Belgium, Israel, the Palestinian Authority, USA, or Australia. Haplotype analysis revealed a common founder for the mutation in Spain, Italy, and the UK. Our data further support the complexity the genetic epidemiology of non-syndromic hearing impairment.