This study characterizes 1,984 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 2005 and 2006 from normally sterile sites in patients with invasive MRSA infection. These isolates represent a convenience sample of all invasive MRSA cases reported as part of the Active Bacterial Core surveillance system in eight states in the United States. The majority of isolates were from blood (83.8%), joints (4.1%), and bone (4.2%). Isolates were characterized by pulsed-field gel electrophoresis (PFGE); SCCmec typing; susceptibility to 15 antimicrobial agents; and PCR analysis of staphylococcal enterotoxin A (SEA) to SEH, toxic shock syndrome toxin 1, and Panton-Valentine leukocidin. Thirteen established PFGE types were recognized among these isolates, although USA100 and USA300 predominated, accounting for 53.2% and 31.4% of the isolates, respectively. As expected, isolates from hospital onset cases were predominantly USA100, whereas those from community-associated cases were predominantly USA300. USA100 isolates were diverse (Simpson's discriminatory index [DI] = 0.924); generally positive only for enterotoxin D (74.5%); and resistant to clindamycin (98.6%), erythromycin (99.0%), and levofloxacin (99.6%), in addition to beta-lactam agents. USA300 isolates were less diverse (DI = 0.566), positive for Panton-Valentine leukocidin (96.3%), and resistant to erythromycin (94.1%) and, less commonly, levofloxacin (54.6%), in addition to beta-lactam agents. This collection provides a reference collection of MRSA isolates associated with invasive disease, collected in 2005 and 2006 in the United States, for future comparison and ongoing studies.