Actinomyces species are uncommon but important causes of invasive infections. The ability of our regional clinical microbiology laboratory to report species-level identification of Actinomyces relied on molecular identification by partial sequencing of the 16S ribosomal gene prior to the implementation of the Vitek MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]) system. We compared the use of the Vitek MS to that of 16S rRNA gene sequencing for reliable species-level identification of invasive infections caused by Actinomyces spp. because limited data had been published for this important genera. A total of 115 cases of Actinomyces spp., either alone or as part of a polymicrobial infection, were diagnosed between 2011 and 2014. Actinomyces spp. were considered the principal pathogen in bloodstream infections (n ؍ 17, 15%), in skin and soft tissue abscesses (n ؍ 25, 22%), and in pulmonary (n ؍ 26, 23%), bone (n ؍ 27, 23%), intraabdominal (n ؍ 16, 14%), and central nervous system (n ؍ 4, 3%) infections. Compared to sequencing and identification from the SmartGene Integrated Database Network System (IDNS), Vitek MS identified 47/115 (41%) isolates to the correct species and 10 (9%) isolates to the correct genus. However, the Vitek MS was unable to provide identification for 43 (37%) isolates while 15 (13%) had discordant results. Phylogenetic analyses of the 16S rRNA sequences demonstrate high diversity in recovered Actinomyces spp. and provide additional information to compare/confirm discordant identifications between MALDI-TOF and 16S rRNA gene sequences. This study highlights the diversity of clinically relevant Actinomyces spp. and provides an important typing comparison. Based on our analysis, 16S rRNA gene sequencing should be used to rapidly identify Actinomyces spp. until MALDI-TOF databases are optimized.A ctinomyces species are uncommon human bacterial pathogens that can cause a wide variety of invasive infections (1). The Actinomyces genus is part of the family Actinomycetaceae, of which there are currently 47 published species, but several novel Actinomyces taxa have recently been described by molecular analyses targeting the ribosomal 16S rRNA gene (2). For example, the Human Oral Microbiome Database (www.homd.org) currently reports many species-level Actinomyces taxa that have not yet been named (3). The pathogenic role of known and novel Actinomyces spp. in contributing alone or as part of polymicrobial flora to human invasive infections is underappreciated because many clinical microbiology laboratories continue to rely on phenotypic methods for identification (4-7). Because Actinomyces spp. grow best under anaerobic conditions and are notoriously fastidious, slow -growing organisms, they may be missed as important pathogens by routine aerobic culture techniques and incubation periods (2,8).Upon microscopic examination, Actinomyces isolates typically appear as Gram-positive bacilli that may be coryneform-like with palisading or branching ...