Overall, 2,337 rectal screening samples (RSSs) were seeded by using the Wasp instrument for automated microbiological processing with five media for detection of extended-spectrum β-lactamase (ESBL): CHROMagar, ChromID, Brilliance, BD Drigalski, and HEGP media. Of 354 RSSs harboring ESBL-producing isolates, 89.3% were found to be positive on all media. Sensitivity and specificity ranged from 95.5 to 98.3% and from 57.9 to 72.3%, respectively. No medium was perfectly ESBL selective, and non-ESBL-producing strains were mainly Enterobacteriaceae overproducing AmpC β-lactamase and nonfermenting Gram-negative bacilli, mostly Pseudomonas aeruginosa.