Sato is a traditional Thai alcohol beverage produced by the fermentation of steamed rice with a microbial starter. The industrial production of sato faces problems with inconsistent quality due to the variability in the microbial community of the starter. Previously, an NP1 starter was selected from 114 starter samples for its abilities to liquefy rice, to ferment and produce ethanol, and to give a good flavour and taste to the resultant sato. Here, we developed a defined starter culture mixture for sato fermentation, according to the composition of the microorganisms identified in the NP1 starter. The oenological parameters during the fermentation with the original NP1 starter and the defined starter culture mixture were compared. The NP1 starter and the alternative starter exhibited similar microbial population dynamics, as determined by conventional cultivation-dependent methods and by cultivation-independent denaturing gradient gel electrophoresis (DGGE) analysis. The profiles of the organic acids, glycerol, and the volatile compounds produced during fermentation were similar. The sato fermented with the two different starters gained similarly high scores of sensory evaluation. Based on these data, we concluded that the defined starter culture mixture has a great potential to become an alternative starter to produce high quality sato with consistency and may facilitate industrial production of sato. The advantages of using the DGGE analysis in combination with the conventional culture method to study the microbial population dynamics during the fermentation process of sato are discussed.