Summary The cellular long-chain fatty acids of 40 strains, representing eight yeast species, were examined by gas Chromatographic analyses on a polar capillary column. Fatty acids were identified by mass spectral analyses of methyl and picolinyl esters. The mean relative percentages of 12 fatty acids were used to differentiate between strains and species. Data were compared by visual examination, analysis of variance, calculation of the index of relationship (R), calculation of the similarity coefficient (So) and stepwise discriminant analysis (SDA). Strains within a species were compared by all methods excluding SDA. The So-values were considered to give a more accurate approximation of the similarity/relatedness between a pair of strains than the R-values. Thirty seven strains had unique fatty acid profiles, confirming the utility of cellular fatty acid analysis as a yeast strain characterisation technique. Differentiation between species was based on SDA. An increase in the number of replicates per strain and the number of variables (fatty acids) improved the classification ability of the technique. While it was possible to differentiate between the majority of species, the data indicated that this was not possible in a number of cases. Saccharomyces cerevisiae could be distinguished from all species studied, except possibly Pachytichospora transvaalensis.