Emission from ruminants has become one of the largest sources of anthropogenic methane emission in China. The structure of the rumen flora has a significant effect on methane production. To establish a more accurate prediction model for methane production, the rumen flora should be one of the most important parameters. The objective of the present study was to investigate the relationship among changes in rumen flora, nutrient levels, and methane production in sheep fed with the diets of different forage-to-concentration ratios, as well as to screen for significantly different dominant genera. Nine rumen-cannulated hybrid sheep were separated into three groups and fed three diets with forage-to-concentration ratios of 50:50, 70:30, and 90:10. Three proportions of the diets were fed according to a 3 × 3 incomplete Latin square, design during three periods of 15d each. The ruminal fluid was collected for real-time polymerase chain reaction (real-time PCR), high-throughput sequencing and in vitro rumen fermentation in a new real-time fermentation system wit. Twenty-two genera were screened, the abundance of which varied linearly with forage-to-concentration ratios and methane production. In addition, during the 12-hour in vitro fermentation, the appearance of peak concentration was delayed by 26-27min with the different structure of rumen bacteria. The fiber-degrading bacteria were positively correlated with this phenomenon, but starch-degrading and protein-degrading bacteria were negative correlated. These results would facilitate macro-control of rumen microorganisms and better management of diets for improved nutrition in ruminants. In addition, our findings would help in screening bacterial genera that are highly correlated with methane production.