Mutations in the gene encoding dysferlin (DYSF; MIM# 603009, 2p13, GenBank NM_003494.2) cause primary dysferlinopathies, which are autosomal recessive muscular dystrophies. DYSF has a large mutational spectrum, and genetic diagnosis is complicated by incomplete mutation detection rates. Recently, novel dysferlin transcripts were characterized by identifying alternative exons 1 of DYSF-v1 (GenBank DQ267935), exon 5a (GenBank DQ976379), and exon 40a (GenBank EF015906). To evaluate the frequency of possible mutations in the newly identified DYSF alternative exons, we screened the corresponding genomic regions for mutations in a cohort of 26 patients, carrying only one mutation undoubtedly considered as disease causing in the 55 canonical DYSF exons. No disease-causing mutation was identified in alternative exons 1 of DYSF-v1, exon 5a, and exon 40a, demonstrating a low frequency of disease-causing mutations in these exons.