The aim of the study was to determine (1) the frequency and type of mutations in the coding region of the GJB2 gene (sequencing), (2) the frequency of splice site mutation IVS1 + 1G > A in the GJB2 gene (multiplex ligation-dependent probe amplification analysis), (3) possible copy number changes in the GJB2, GJB3, GJB6, and WFS1 genes (multiplex ligation-dependent probe amplification analysis), and (4) the frequency of del(GJB6-D13S1830) in the GJB6 gene in 58 unrelated patients with nonsyndromic hearing loss from Croatia.