Rapid diagnosis of infections caused by carbapenem-resistant Enterobacteriaceae (CRE) is crucial for proper treatment and infection control. The Xpert® Carba-R assay is a qualitative multiplex real-time PCR method that qualitatively detects and differentiates five common carbapenemase genes (blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP) directly from rectal swabs or purified colonies within an approximately one hour. We performed a multicenter evaluation of the investigational use of the Carba-R assay for detection and differentiation of carbapenemase genes from sputum specimens in patients with a clinical diagnosis of pneumonia. The intra- and inter-assay coefficients of variation values for the Carba-R assay were 0.2% to 2.0% and 1.4% to 2.3%, respectively. A total of 301 sputum specimens were collected and tested. When compared to bacterial culture followed by PCR identification of resistance genes from colonies, the Carba-R assay reduced turnaround time from 56-84 hours to less than two hours. Carbapenemase genes were detected by the Carba-R assay in Klebsiella pneumoniae (n=236), Escherichia coli (n=22), Enterobacter cloacae (n=23), K. oxytoca (n=8), Serratia marcescens (n=6), Citrobacter freundii (n=4), and Klebsiella aerogenes (n=2). The Carba-R assay detected 112 blaKPC (33.5%), 70 blaNDM (21.0%), 8 blaIMP (2.4%), 2 blaVIM (0.6%), with positive percent agreement, negative percent agreement and concordance rates of 92.9%, 86.7%, and 88.3% for the dominant blaKPC and 85.0%, 87.8%, and 87.4% for the blaNDM genes. Neither method detected the blaOXA-48 carbapenemase gene. The convenient, rapid and simple characteristics of the Xpert® Carba-R assay make it a potential tool for CRE detection and identification directly in sputum specimens. Copyright © 2020 American Society for Microbiology.