Accurate species determination for anaerobes from blood culture bottles has become increasingly important with the reemergence of anaerobic bacteremia and prevalence of multiple-drug-resistant microorganisms. Our knowledge of the taxonomical diversity of anaerobes that cause bloodstream infections is extremely limited, because identification historically has relied on conventional methods. Over a 5-year period, we profiled anaerobic bacteremia at a large tertiary care hospital with 16S rRNA gene sequencing to gain a better understanding of the taxonomical diversity of the bacteria. Of 316 isolates, 16S rRNA gene sequencing and phylogenetic analysis identified 316 (100%) to the genus or taxonomical group level and 289 (91%) to the species level. Conventional methods identified 279 (88%) to the genus level and 208 (66%) to the species level; 75 (24%) were misidentified at the species level, and 33 (10%) results were inconclusive. High intragenus variability was observed for Bacteroides and Clostridium species, and high intraspecies variability was observed for Bacteroides thetaiotaomicron and Fusobacterium nucleatum. Sequence-based identification has potential benefits in comparison to conventional methods, because it more accurately characterizes anaerobes within taxonomically related clusters and thereby may enable better correlation with specific clinical syndromes and antibiotic resistance patterns.