ABSTRACTAntimicrobial susceptibility patterns of 112 clinical isolates, 28 type strains, and 9 reference strains ofNocardiawere determined using the Sensititre Rapmyco microdilution panel (Thermo Fisher, Inc.). Isolates were identified by highly discriminatory multilocus sequence analysis and were chosen to represent the diversity of species recovered from clinical specimens in Ontario, Canada. Susceptibility to the most commonly used drug, trimethoprim-sulfamethoxazole, was observed in 97% of isolates. Linezolid and amikacin were also highly effective; 100% and 99% of all isolates demonstrated a susceptible phenotype. For the remaining antimicrobials, resistance was species specific with isolates ofNocardia otitidiscaviarum,N. brasiliensis,N. abscessuscomplex,N. novacomplex,N. transvalensiscomplex,N. farcinica, andN. cyriacigeorgicadisplaying the traditional characteristic drug pattern types. In addition, the antimicrobial susceptibility profiles of a variety of rarely encountered species isolated from clinical specimens are reported for the first time and were categorized into four additional drug pattern types. Finally, MICs for the control strainsN. novaATCC BAA-2227,N. asteroidesATCC 19247T, andN. farcinicaATCC 23826 were robustly determined to demonstrate method reproducibility and suitability of the commercial Sensititre Rapmyco panel for antimicrobial susceptibility testing ofNocardiaspp. isolated from clinical specimens. The reported values will facilitate quality control and standardization among laboratories.