Abstract: Objective To explore the genetic mutation in neonates who failed to pass hearing screening. Methods A total of 111 cases of neonates who failed to pass hearing screening and were confirmed sensorineural deafness by auditory brainstem evoked potential (ABR) were randomly selected. The heel blood was collected and DNA was extracted. GJB2, SLC26A4, and 11 mutation hotspots in mitochondria gene 12SrRNA were tested. The relationship between degree of hearing loss and gene mutation was analyzed. Results In 111 neonates, mutation in deafness gene were found in 24 cases (21.6%) . Among them 14 cases (12.6%) had GJB2 gene mutation including 5 cases of 235delC single heterozygous mutation, 5 cases of 235delC, and 1 case each of 299_300delAT compound heterozygous mutation, 235delC homozygous mutation, 299_300delAT single heterozygous mutation, 176_191del16 and 235delC compound heterozygous mutation, and 299_300delAT and 508_511dupAACG compound heterozygous mutation respectively. Ten cases (9.0%) had SLC26A4 gene mutation including 2 cases of IVS7-2A ＞ G single heterozygous mutation, 3 cases of 1226G ＞ A single heterozygous mutation, 2 cases of 2168A ＞ G single heterozygous mutation, and 3 cases of IVS7-2A ＞ G and 2168A ＞ G compound heterozygous mutation. Mitochondrial gene mutations were not detected. Conclusions Deafness gene mutation is detected in more than 1/5 neonates who failed to pass newborn hearing screening. GJB2 gene mutation is the most commons. The implementation of hotspots deafness gene detection can improve the diagnostic rate of deafness.