SummaryAlthough typically cosseted in the laboratory with constant temperatures and plentiful nutrients, microbes are frequently exposed to much more stressful conditions in their natural environments where survival and competitive fitness depend upon both growth rate when conditions are favourable and on persistence in a viable and recoverable state when they are not. In order to determine the role of genetic heterogeneity in environmental fitness we present a novel approach that combines the power of fluorescence‐activated cell sorting with barcode microarray analysis and apply this to determining the importance of every gene in the Saccharomyces cerevisiae genome in a high‐throughput, genome‐wide fitness screen. We have grown > 6000 heterozygous mutants together and exposed them to a starvation stress before using fluorescence‐activated cell sorting to identify and isolate those individual cells that have not survived the stress applied. Barcode array analysis of the sorted and total populations reveals the importance of cellular recycling mechanisms (autophagy, pexophagy and ribosome breakdown) in maintaining cell viability during starvation and provides compelling evidence for an important role for fatty acid degradation in maintaining viability. In addition, we have developed a semi‐batch fermentor system that is a more realistic model of environmental fitness than either batch or chemostat culture. Barcode array analysis revealed that arginine biosynthesis was important for fitness in semi‐batch culture and modelling of this regime showed that rapid emergence from lag phase led to greatly increased fitness. One hundred and twenty‐five strains with deletions in unclassified proteins were identified as being over‐represented in the sorted fraction, while 27 unclassified proteins caused a haploinsufficient phenotype in semi‐batch culture. These methods thus provide a screen to identifying other genes and pathways that have a role in maintaining cell viability.