Molecular Genetic Diversity and DNA Diagnostics of Hereditary Spastic Paraplegia Abstract—Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurodegenerative disorders predominantly characterized by damage to the pyramidal tract. The major, single symptom of HSP is progressive weakness and spasticity of lower extremities, which ultimately leads to difficulties in walking. HSP has various types of inheritance and clinical characteristics. The development of molecular genetics and the emergence of high-throughput sequencing techniques, such as New Generation Sequencing (NGS) in particular, revealed a pronounced genetic heterogeneity of HSP. Currently, about 80 genetic loci for HSP are designated as spastic paraplegia genes (SPGs) (with numbering according to the mapping history), and the vast majority of causative genes within loci have been identified. The most frequent autosomal dominant forms of HSP are SPG4 and SPG3; SPG11, SPG15, SPG7 are common autosomal recessive HSPs. The mechanisms of HSP molecular pathogenesis are variable and encompassing defects of neuronal membrane transport and the disturbance of myelination processes, lipid metabolism, mitochondrial dysfunctions, etc. The differential diagnosis for HSPs is difficult due to the existence of many geno- and phenocopies within the other neuropathologies that can be partially distinguished by neuroimaging methods. The only method for the precise diagnostics of HSP and the number of genomic copies is DNA diagnostics—the search for mutations in individual genes and/or the analysis of many genes simultaneously by NGS methods (gene panel, whole-exome sequencing, and whole-genome sequencing). Such DNA diagnostics opens up the opportunity to perform prenatal or preimplantation analysis of the fetus for families carrying HSP mutations to predict the risk of HSP inheritance. Currently, HSP treatment is directed against individual symptoms (antispastic drugs, etc.), and the discovery of the pathogenic action of different genes requires the development of new therapeutic approaches.