Abstract: Objective　To explore the clinical and genetic characteristics of nemaline myopathy induced by NEB gene mutation. Method　The clinical data of suspected nemaline myopathy patients and their family members in two families and results of gene sequencing using targeted gene capture second-generation sequencing were retrospective analyzed. Results　The age at onset of proband in both families was around 2 years old. They went to see a doctor because of lower limb muscle weakness and unstable walking. The children had low muscle tension with grade IV limb muscle strength. The electromyography showed no myogenic damage. Serum creatine kinase (CK) level was slightly elevated. Complex heterozygous mutations of c.2227C>T and c.16972G>T in NEB gene which were inherited from parents respectively, were found in one patient by gene testing, and the elder brother was a carrier of c.5971C>T heterozygous mutation. In the other patient, there were complex heterozygous mutations of c.6388G> c and c.17044A>T in the NEB gene which were inherited from the parents respectively. Most of their family members were carriers. Conclusion In patients with myasthenia and hypotonia clinicians should be alert to the possibility of congenital nemaline myopathy. Gene testing is helpful for diagnosis. Both c.5971C>T and c.17044A>T in NEB gene are newly discovered mutation site.