Abstract DNAJB6 myopathy is an autosomal dominant myopathy characterized by middle-aged onset of lower limb muscle weakness and presence of rimmed-vacuoles (RVs) and cytoplasmic inclusions (CIs) on muscle pathology. DNAJB6 is a member of DNAJ (HSP40) family, having roles in anti-protein aggregation and growth inhibition of cardiomyocytes. So far, only three different mutations have been identified in 11 unrelated families. To identify and characterize DNAJB6 myopathy in Japanese cohort. Patients and methods: We searched for DNAJB6 mutations by direct sequencing in 103 LGMD families and 89 families with RVs and/or CIs on muscle pathology. Clinical and pathological characteristics were analyzed. We found four families with DNAJB6 myopathy. One family had a novel p.F93I substitution in the G/F domain and three had the same p.F93L mutation as previously reported. The patient carrying p.F93I substitution was a 36-year-old male and manifested slowly progressive lower limb weakness from 30s. No cardiac or brain involvement was seen. His father and elder brother showed similar clinical features. Serum CK levels was elevated to 1044IU/L and EMG showed myogenic pattern. Patients carrying the p.F93L mutation also showed adult onset mild myopathy. Muscle images commonly showed preferential involvement of posterior lower limb muscles with preserved rectus femoris (RF) and tibialis anterior (TA) muscles. Skeletal muscles showed myopathic changes with a few fibers with RVs and small cytoplasmic/nuclear inclusions. On immunohistochemistry, accumulations of DNAJB6, TDP43 and the other interacted proteins were seen. Four Japanese families with DNAJB6 myopathy were identified including one with a novel mutation. All patients showed similar clinical and pathological findings. Preservation of RF and TA was characteristic. The G/F domain of DNAJB6 may have important roles in skeletal muscle.