The LEADER Program monitors the in vitro activity of linezolid in sampled U.S. medical centers using reference broth microdilution methods with supporting molecular investigations in a central laboratory design. This report summarizes data obtained in 2009, the 6th consecutive year of this longitudinal study. A total of 6,414 isolates from 56 medical centers in all nine Census regions across the United States participated in 2009. For the six leading species/groups, the following linezolid MIC(90) values were observed: Staphylococcus aureus, 2 μg/ml; coagulase-negative staphylococci (CoNS), 1 μg/ml; Enterococcus spp., 2 μg/ml; Streptococcus pneumoniae, 1 μg/ml; viridans group streptococci, 1 μg/ml; and beta-hemolytic streptococci, 1 μg/ml. Linezolid resistance was only 0.34% overall, with no evidence of significant increase in the LEADER Program since 2006. The predominant linezolid resistant mechanism found was a G2576T mutation in the 23S rRNA. L3/L4 riboprotein mutations were also found. The mobile multidrug-resistant cfr gene was found in four strains (two S. aureus strains and one strain each of S. epidermidis and S. capitis) from four different states, suggesting persistence but a lack of dissemination. Linezolid continues to exhibit excellent activity and spectrum, and this study documents the need for continued monitoring of emerging mechanisms of resistance over a wide geographic area.