Next generation sequencing (NGS) is having a tremendous impact on our knowledge of different aspects of biology. It can be also very powerful to study patients with heterogeneous genetic conditions, like muscular dystrophies. First, to identify new genes using “exome resequencing”. Second, to diagnose mutations in all the known causative genes, when used as targeted approach. Third to obtain a knowledge of the impact of mutations on mRNA expression and splicing in diseased muscle. We used NGS to identify new genes by whole exome sequencing. We sequenced the whole exome of four family members with LGMD1F separated by up to eleven meioses and identified a single shared novel heterozygous frame-shift variant. This causes a nonstop change in the Transportin 3 (TNPO3) gene that encodes a member of the importin-β super-family. To reach the second task, we first recruited 160 familial cases of nonspecific limb-girdle muscular dystrophies with apparent autosomal inheritance. All DNA samples were first enriched for 486,480bp, covering 2447 exons of 98 genes by using the Haloplex technology with the use of barcodes. We the performed pooled NGS of all samples and identified a number of mutations, then verified by Sanger sequencing. Cases were also studied by the Agilent MotorChip CGH array version 3.0 to identify deletions or duplications. Finally, in selected cases, we performed the RNA-Seq starting from a muscle biopsy sample. We converted mRNA to cDNA and purified it by a customized SureSelect Target Enrichment System, focused on the same 98 mRNAs. The probes had a 4× coverage with a total target of 1.41Mb of sequences/sample. These cDNAs were sequenced using barcodes trying to obtain an average sequencing coverage of at least 100×. Our results confirm that there is a very high genetic heterogeneity in muscular dystrophies and that NGS-based DNA and RNA testing are ready for diagnostic use.