Central core disease, one of the most common congenital myopathies in humans, has been linked to mutations in the RYR1 gene encoding the Ca2+ release channel of the sarcoplasmic reticulum (RyR1). Functional analyses showed that disease-associated RYR1 mutations led to impairment of skeletal muscle Ca2+ homeostasis, however, thorough understanding of the molecular mechanisms underlying central core disease and other RyR1-related conditions is still lacking. We screened by sequencing the complete RYR1 transcripts in ten unrelated patients with central core disease and identified five novel, p.M4640R, p.L4647P, p.F4808L, p.D4918N and p.F4941C, and four recurrent mutations. Four of the novel mutations involved amino acid residues that were positioned within putative transmembrane segments of the RyR1. The pathogenic character of the identified mutations was demonstrated by bioinformatic analyses and by the in vitro functional studies in HEK293 cells and RYR1-null (dyspedic) myotubes.