Canola mesophyll protoplasts are routinely used as an explant source to generate transgenic plants. Methods to enrich for sub-populations of mesophyll protoplasts that are maximally responsive to direct DNA delivery and whole plants regeneration would be beneficial. Here we report the deployment of FACS based assays and sorting methods to investigate heterogeneity in canola mesophyll protoplasts. Freshly isolated protoplasts suspensions were assayed using forward and side scatter properties to characterise sub-populations based on size and chloroplast content. Four significant populations were identified. Sub-population 1, comprising chloroplast-rich cells of 40-50 µm in size, were most responsive to PEG mediated DNA delivery. To further investigate the sub-populations, a two-step approach involving FACS sorting and confocal microscopy, and a single step approach using image flow cytometry was deployed. Results obtained from each approach aided the development of processes to enrich for sub-population 1. Our approach resulted in a two-fold increase in transfection efficiency, and illustrates the use of flow cytometry based tools (FACS & Image flow cytometry) to fast track the development of improved purification procedures in the routine workflows.