This study was conducted to evaluate the feed-nutritional value of spent mushroom substrates(SMS) as a roughage source for ruminants through in vitro and in situ experiments. The SMS was classified into a roughage source with high fiber(NDF 64～78%) and low protein(CP 7～11%). The chemical composition of SMS was affected mainly by the primary culture ingredient rather than mushroom species. Compared with sawdust-SMS, cotton waste-SMS contained less(P<0.05) NDF and more(P<0.05) nonfibrous carbohydrate and ash(P<0.05). In vitro DM and NDF disappearances were high in the order of corn cob-, cotton waste-, and sawdust-SMS, in situ DM, NDF and ADF disappearances at 24hr incubation also showed the same pattern with in vitro trials. Compared with sawdust-SMS, cotton waste-SMS had higher digestible fractions and lower non-digestible fractions of NDF and ADF(P<0.05), resulting in higher in situ DM and NDF disappearances(P<0.05) and higher ruminal degradability(P<0.05). Therefore, the preferential use of cotton waste-SMS to sawdust-SMS is recommended as a roughage source for ruminants.