The present study evaluated the influence of rumen inocula of different breeds on in vitro fermentation with forage and concentrate substrates. An in vitro was conducted under a 2×2 factorial arrangement with two breeds (Jersey and Holstein steers) and two feed substrates (forage and concentrate) as factors. Three Jersey and 3 Holstein steers were used for the source of in vitro inocula. Metataxonomic analysis of donor rumen fluids showed that Firmicutes was more abundant in Jersey, while Bacteroidetes in Holstein steers. In vitro ㏗ was lower in the fermented inocula of Jersey steers and in the concentrate substrate (p<0.05). After 24h, higher gas production, dry matter, and neutral detergent fiber degradability, and total volatile fatty acids concentration were noted in concentrate substrate (p<0.05). After 24h, inocula of Jersey steers had higher methane and ammonia-nitrogen (p<0.05). After 24h, fermented inocula of Holstein steers produced higher propionate (p<0.05). Conversely, in vitro butyrate production was higher in the fermented inocula from Jersey steers (p=0.072) and in those with concentrate substrate (p<0.05). After 24h, the total bacterial population (log10 c opies) was h igher in t he fermented inocula received from Jersey steers and in the concentrate substrate whereas, Fibrobacter succinogenes and Ruminococcus flavefaciens population were higher (p<0.05) only in the concentrate substrate. Overall results suggest that rumen inocula of different donors influence in vitro fermentation either with forage or concentrate substrates.