Summary This study was carried out to examine the possible effect of fresh and frozen pre-fermented juice (PFJ) on the fermentation quality, nutritive value and in vitro gas production parameters of alfalfa (Medicago sativa L.) silage. Barley (B), wheat (W) and grass herbage (G) were used to prepare the PFJs. Both fresh (PFJ-B, PFJ-W and PFJ-G) and frozen (PFJ-B F , PFJ-W F and PFJ-G F ) PFJs were investigated. Frozen PFJs were prepared by freezing fresh PFJs at -22 o C with 20% glycerol (v/v). Treatments of alfalfa silage consist of (1) control (untreated alfalfa silage); (2) silage treated with PFJ-B; (3) silage treated with PFJ-W; (4) silage treated with PFJ-G; (5) silage treated with PFJ-B F ; (6) silage treated with PFJ-W F and (7) silage treated with PFJ-G F . Each treatment had five replicate glass jars and replicates were ensiled in 1.0 L jars. Silages treated with fresh and frozen PFJs, regardless of plant material, had better fermentation quality than the control silage in terms of lower pH, butyric acid (BA) and ammonia nitrogen (NH 3 -N) concentrations. Additionally, treated silages with fresh PFJs had higher lactic acid (LA) concentration (P<0.05) and in vitro organic matter digestibility (IVOMD), metabolizable energy (ME) content and gas production values (P<0.05) than control silage. According to results of this study, PFJ treatments increased the nutritive value, fermentation quality, IVOMD, ME content and gas production values of alfalfa silage.