ABSTRACT We evaluated whether the Bruker Biotyper matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system provides accurate species-level identifications of 147 isolates of aerobically growing Gram-positive rods (GPRs). The bacterial isolates included Nocardia ( n = 74), Listeria ( n = 39), Kocuria ( n = 15), Rhodococcus ( n = 10), Gordonia ( n = 7), and Tsukamurella ( n = 2) species, which had all been identified by conventional methods, molecular methods, or both. In total, 89.7% of Listeria monocytogenes , 80% of Rhodococcus species, 26.7% of Kocuria species, and 14.9% of Nocardia species ( n = 11, all N. nova and N. otitidiscaviarum ) were correctly identified to the species level (score values, ≥2.0). A clustering analysis of spectra generated by the Bruker Biotyper identified six clusters of Nocardia species, i.e., cluster 1 ( N. cyriacigeorgica ), cluster 2 ( N. brasiliensis ), cluster 3 ( N. farcinica ), cluster 4 ( N. puris ), cluster 5 ( N. asiatica ), and cluster 6 ( N. beijingensis ), based on the six peaks generated by ClinProTools with the genetic algorithm, i.e., m/z 2,774.477 (cluster 1), m/z 5,389.792 (cluster 2), m/z 6,505.720 (cluster 3), m/z 5,428.795 (cluster 4), m/z 6,525.326 (cluster 5), and m/z 16,085.216 (cluster 6). Two clusters of L. monocytogenes spectra were also found according to the five peaks, i.e., m/z 5,594.85, m/z 6,184.39, and m/z 11,187.31, for cluster 1 (serotype 1/2a) and m/z 5,601.21 and m/z 11,199.33 for cluster 2 (serotypes 1/2b and 4b). The Bruker Biotyper system was unable to accurately identify Nocardia (except for N. nova and N. otitidiscaviarum ), Tsukamurella , or Gordonia species. Continuous expansion of the MALDI-TOF MS databases to include more GPRs is necessary.