Objective To explore the association between rare HSPB1 variants and amyotrophic lateral sclerosis (ALS). Methods We performed next-generation sequencing for 166 Chinese ALS patients to screen for possible pathogenic rare variants of HSPB1. The control individuals were obtained from 1000 Genome Project and an in-house whole-exome sequencing database. The Sequence Kernel Association Test (SKAT) and the SKAT-optimal test (SKAT-O) were used to identify the association between rare HSPB1 variants and ALS. Results We identified 3 possible pathogenic rare variants of HSPB1 (all were missenses), including c.379C>T (p.R127W), c.446A>C (p.D149A) and c.451A>C (p.T151P). Compared with 1000 Genome Project, SKAT p=3.61×10-7 and SKAT-O p=1.62×10-6 ; while compared with the in-house database, SKAT p=9.99×10-4 , SKAT-O p=1.80×10- 3 . We analyzed the phenotypes of rare HSPB1 variant carriers and found no specific clinical characteristics associated with these variants. Conclusion Rare variants of HSPB1 are probably associated with the pathogenesis of ALS.