DFNB89 is a novel autosomal recessive nonsyndromic hearing impairment (ARNSHI) locus that was mapped to 16q21-q23.2. Linkage to the region was established by carrying out genome-wide linkage scans in two unrelated, consanguineous Pakistani families segregating ARNSHI. The maximum multipoint LOD score is 9.7 for both families and for each family, a significant maximum LOD score of 6.0 and 3.7 were obtained. The 3-unit support interval and the region of homozygosity for the two families extend from rs717293 (chr16: 62.1 Mb) to rs728929 (chr16: 78.2 Mb) and contain 16.1 Mb of sequence. A total of 146 genes are within the DFNB89 interval. Eight candidate genes, CALB2, CDH1, CDH3, CDH11, HAS3, NOB1, PLEKHG4 and SMPD3, were sequenced, but no potentially causal variants were discovered. DFNB89 is the second ARNSHI locus mapped to chromosome 16.