Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurodegenerative diseases characterized by progressive weakness and spasticity of lower limbs. To clarify the genetic spectrum and improve the diagnosis of HSP patients, targeted next-generation sequencing (NGS) was applied to detect the culprit genes in 55 Chinese HSP pedigrees. The classification of novel variants was based on the American College of Medical Genetics and Genomics (ACMG) standards and guidelines. Patients remaining negative following targeted NGS were further screened for gross deletions/duplications by multiplex ligation-dependent probe amplification (MLPA). We made a genetic diagnosis in 61.8% (34/55) of families and identified 33 mutations, including 14 known mutations and 19 novel mutations. Of them, one was de novo mutation (NIPA1: c.316G>A). SPAST mutations (22/39, 56.4%) are the most common in Chinese AD-HSP followed by ATL1 (4/39, 10.3%). Moreover, we identified the third BSCL2 mutation (c.1309G>C) related to HSP by further functional studies and first reported the KIF1A mutation (c.304G>A) in China. Our findings broaden the genetic spectrum of HSP and improve the diagnosis of HSP patients. These results demonstrate the efficiency of targeted NGS to make a more rapid and precise diagnosis in patients with clinically suspected HSP. We made a genetic diagnosis in 61.8% of families and identified 33 mutations. SPAST mutations are the most common in Chinese AD-HSP followed by ATL1. Our findings broaden the genetic spectrum and improve the diagnosis of HSP.