Ecological interactions between different species of yeasts have been observed and described extensively, but the mechanisms of interaction remain poorly understood. A hindrance to the characterization of multispecies yeast ecosystems is the lack of accurate methods for rapid real-time analysis of population dynamics in synthetic multispecies consortia. Here, we sought to accelerate and improve the sensitivity of ecological modelling and characterization of a synthetic yeast ecosystem by developing a flow cytometry-based method that tracks and sorts fluorescently tagged individual yeast species in real time during growth in model multispecies consortia. A protocol for integrative genetic modification of non-conventional yeasts was developed. The application of the method was demonstrated in a model four-species synthetic wine-yeast ecosystem that consisted of species commonly isolated from natural wine fermentations. The data show that this method allows for rapid generation of meaningful ecological data that contributes to our understanding of multispecies synthetic yeast ecosystems. Furthermore, interspecies interactions have been shown to impact the evolution of yeasts in natural ecosystems, and this platform will provide an ideal tool to better evaluate the impact of biotic selection pressures.