Objective: To investigate the mutations of SLC26A4 gene and the relevant phenotype in Chinese sporadic nonsyndromic hearing-impaired children. Methods: 195 Chinese sporadic nonsyndromic hearing-impaired children were subjected to microarray-based mutation detection for 9 hot spot mutations in four of the most common deafness-related genes (GJB2, SLC26A4, GJB3, and 12s rRNA). Subsequently, twenty-one patients with one SLC26A4 mutation detected by microarray were subjected to sequencing analysis of the whole SLC26A4 coding region and the splice sites in order to identify the second mutant allele. The inner ear malformation and hearing loss level were compared among different genotypes. Results: The incidence of genetic mutations was found to be 43.59% (85/195) in this patient group using CapitalBio Deafness Gene Mutation Detection Array Kit. A total of 34 children (17.44%) were found carrying the mutant SLC26A4 sequences. Thirteen (6.67%) children carried two mutant alleles of SLC26A4 and 21(10.77%) children carried one mutant allele of SLC26A4. After the application of subsequent sequencing analysis, 13 mutational variants including 4 novel variants, two missense (p.D661G, p.N457D), one splice site mutation (IVS15+1G>A) and one frameshift mutation (624_632del9in-sACITGGC), were identified in SLC26A4 gene in 15 of the 21 previously monoallelic patients. No second mutation was identified in the remaining 6 children. Biallelic mutations of SLC26A4 were identified in 20 of 21 children with enlarged vestibular aqueduct. Conclusions: Our results demonstrated that genetic factors were important causes for sporadic nonsyndromic hearing loss in Chinese pediatric cases. Mutation of SLC26A4 is one of the major genetic causes in nonsyndromic hearing loss with inner ear malformation. IVS7-2A>G, 2168A>G and 1229C>T were the most frequent mutations identified in our studies. The combination of microarray testing and sequencing analysis is a useful and high-throughput method for the diagnosis of genetic hearing loss. (C) 2012 Elsevier Ireland Ltd. All rights reserved.