For any genetic based disease, obtaining a definitive diagnosis is critical for proper disease management, family planning, and participation in clinical trials. This process can be challenging for dysferlinopathy due to the significant clinical overlap between the 30+ subtypes of limb-girdle muscular dystrophy (LGMD), of which dysferlinopathy is a part, and the large number of variants of unknown significance (VUS) that are identified in DYSF. We performed RNA-Seq in 79 individuals with a clinical or genetic suspicion of dysferlinopathy or a closely related LGMD and used the information obtained to evaluate all 113 DYSF variants according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. This evaluation lead to the identification of 11 novel DYSF variants and the classification of 89 DYSF variants as pathogenic or likely pathogenic, 8 as likely benign, while 16 variants remained VUSs. By the end of the study, 61 of the 79 cases had a definitive diagnosis of dysferlinopathy which was a 48% increase in the diagnostic yield over the diagnosis rate prior to the onset of the study. This data shows the ability of RNA-Seq to aid in variant pathogenicity classification and diagnosis of dysferlinopathy. The study also revealed other diagnostic techniques such as copy number variation analysis, repeat DNA sequencing, and evaluation of dysferlin protein levels that could be used instead of or in addition to RNA-Seq to finalize the diagnosis. Therefore, these types of analyses should be considered by clinicians when initial genetic sequencing does not provide enough information to allow for a definitive diagnosis of dysferlinopathy.