Spastic paraplegia type 64 (SPG64; OMIM 615683) is a complicated form of hereditary spastic paraplegia (HSP) recently identified in individuals diagnosed with suspected neurodegenerative disease. Affected patients carry homozygous mutations in the ectonucleoside triphosphate diphosphohydrolase 1 gene (ENTPD1). Although they share common characteristics, affected individuals show slight discrepancies in some clinical aspects. At present, only two different cases of SPG64 have been diagnosed. More findings of genetic variation would be helpful to better understand the effect of mutations in the ENTPD1 gene on the neurological condition of affected individuals. In this study, we examined a family with an individual diagnosed with suspected HSP based on clinical findings. DNA samples from the proband, her affected sister, and both parents were analyzed using next-generation sequencing. We used an in-house automated pipeline to detect potential neuromuscular disease-causing variants. Variants were confirmed by Sanger sequencing. After cosegregation analysis, the variant NM_001776.5:c.401T>G (p.M134R) of the ENTPD1 gene was identified as a novel missense mutation linked to the phenotype of SPG64 in the proband and her sister, who showed similar and distinct clinical features compared with the two cases previously described in the literature.