Hereditary spherocytosis (HS) is an inherited disorder characterized by anemia, splenomegaly, and spherical-shaped erythrocytes, caused by mutations in erythrocyte membrane Protein Genes (ANK1,SPTB,SLC4A1,SPTA1, andEPB42). We investigated molecular spectrum and genotype-phenotype correlation in HS patients in Hubei province, central China. Twenty-three patients with HS were included. A next-generation sequencing (NGS) panel targetingANK1,SPTB,SLC4A1,SPTA1, andEPB42genes was used to screen potential variants. Sanger sequencing was applied to validate variants. Of the twenty-three patients, thirteen patients carriedANK1variants, and ten patients harboredSPTBvariants, including ten non-sense, six indel, four splice site, one start-loss, and one missense variant. Four out of twenty-two variants in our study were known, and eighteen variants were novel. MostANK1andSPTBvariants were indel (5/12) or non-sense (7/10), respectively. Family member analysis in thirteen families showed that six variants werede novo. Variable expressivities were observed in a pair of twins withANK1c.341C > T variant, and two unrelated patients both carriedANK1c.2T > A variant. Genotype-phenotype analysis found no significant difference betweenANK1andSPTBregarding the levels of Hb, RBC, MCV, MCH, and MCHC. However, variants in the ANK1 death domain were associated with lower levels of MCV and MCH compared to other ANK1 domains. In conclusion, NGS is a fast way to provide a molecular HS diagnosis. We also identified unique genetic and clinical characteristics of patients with HS in Hubei Province, China. However, a large sample size is needed to further investigate the genotype-phenotype correlation.