Wilson's disease (WD) is an autosomal recessive inheritance disorder of copper metabolism due to mutations in the ATP7B gene. The distribution of ATP7B gene mutations is diverse in different population. This study aimed to examine the genotypes of the ATP7B mutant alleles in WD patients from Southern China. Genomic DNA was extracted from 103 WD patients and 60 healthy patients. Mutations were screened and detected by DNA sequencing. A total of 51 different ATP7B mutations were identified in WD patients, including six homozygous, 51 compound heterozygous, and 39 single heterozygotes. Three mutations were found to be novel, including one missense mutation (c.2549C>T) and two frameshift mutations (c.3851_3876del and c.1057delC). The most frequent mutations are Arg778Leu (18.93%), Ile1148Thr (8.74%), and Pro992Leu (4.37%). Different from the published results of early studies, Ile1148Thr was found to be the second common mutation in our cohort. The highest mutation detection rate was on exon 8 (43.69%), followed by exon 16 (24.27%), and exon 12 (17.48%). The total mutation detection rate on exon 8, 12, and 16 was 85.44%. No ATP7B gene mutation was found in healthy patients. In conclusion, we identified three novel mutations and Ile1148Thr as another hotspot mutation in WD patients from Southern China. Most of the mutations can be detected by screening exon 8, 12, and 16. Our research has further enriched the mutation spectrum of the ATP7B gene in Chinese and may help to develop genetic screening strategies of WD.