We evaluated the ability of the yeast killer system to differentiate members belonging to the Nocardia asteroides complex (Nocardia asteroides, Nocardia farcinica, and Nocardia nova). Nocardia strains were selected randomly from clinical isolates. Type strains of each Nocardia species and recognized killer yeasts were taken from different collections. A clear area of inhibition surrounding the yeast cells demonstrated a positive killer effect on Nocardia spp. Two yeast strains, Pichia mrakii (K9) and Pichia lynferdii (K76), showed different killer activities against each Nocardia species. The group N. asteroides was identified as K9+ K76-, the group N. farcinica was identified as K9- K76-, and the group N. nova was identified as K9+ K76+. The three killer identifications correlated with specific taxonomic groups determined by using classical methods. The yeast killer system may be a useful means for identifying organisms within the N. asteroides complex.