Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM), and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233,140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, since we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA, but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a "nonstop" mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤ 20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.