The SPAST gene has a major role in hereditary spastic paraplegias (HSPs). This is the first report mapping characteristics of the SPAST gene in a large cohort of Czech HSP patients. All 17 coding exons of the SPAST gene were Sanger sequenced in 327 patients from 263 independent families with suspected uncomplicated HSP. The selected 126 independent patients, without mutation in the SPAST gene after Sanger sequencing, were subsequently tested by Multiplex Ligation-dependent Probe Amplification (MLPA) assay for large deletions or copy number variations affecting the SPAST gene. Among the 263 independent patients, 35 different, small mutations in 44 patients were found. Twenty-one mutations are novel with the majority of frameshift mutations. Seven mutations were found in more than one family. The age at onset ranged between preschool childhood and the fifth decade with inter- and intra-familiar differences. SPAST small mutations were detected in 16.7% (44/263) of independent tested patients. Mutations in the SPAST gene were found more frequently in familial cases (with affected relatives). Mutation were found in 31.9% (29/91 familial tested) in the familial patient group, whereas in the sporadic patient group, mutations were found in only 4.7% of cases (5/106 sporadic cases). Among SPAST-positive patients, 65.9% (29/44) were familial but only 11.4% (5/44) were sporadic. MLPA testing revealed four large deletions in four independent patients, all in familial-positive cases. Mutations in the SPAST gene are 5.8 × more frequent in familial than in sporadic cases. Large deletions were found only in familial patients. Diagnostic testing of the SPAST gene is useful only in positive family history patients not in sporadic cases.