The aim of this study was to investigate the characteristics of multi-starter fermentation with Chinese indigenous yeast and commercial strains. UCD522K and UCD2610K carrying the kanamycin resistance gene （KanMX） were constructed using commercial S. cerevisiae UCD522 and UCD2610 as the original strains. The fermentation rates and yeast population were not affected by the resistance marker during the fermentations under two levels （55， 433 mg N/L） of yeast assimilable nitrogen （YAN） concentrations. On this basis， the characteristics of mixed fermentation （inoculation ratio 1∶1） with indigenous yeast NX11424 and UCD522K or and UCD2610K were studied respectively. The results showed that compared with pure fermentation， mixed fermentation did not change the fermentation process， and their fermentation rates were not significantly different under high YAN level. The rate of mixed fermentation was between each of pure fermentation under low YAN level. And it was also closer to the fast fermentation strain. The amount of each yeast in the mixed fermentation was influenced by the concentration of YAN. NX11424 continued predominance during the mixed fermentation with UCD522K at high YAN level， while it showed a ratio of 1 ∶ 1 throughout the fermentation under low YAN level. In the mixed fermentation with UCD2610K， the ratio of NX11424 to UCD2610K was maintained at 1 ∶ 1 regardless of YAN concentrations. A kanamycin resistance marker-aided approach with higher screen efficiency was successfully applied in the multi-starter fermentation of wine-like condition in this work， realizing the rapid and sensitive monitor of the number of different S. cerevisiae strains during fermentation. This study also found that indigenous Chinese strain NX11424 could dominate in the wine-making environment with strong fermenting performance and killer commercial yeasts， providing data support for making wines with more particular flavor.