OBJECTIVE To assess knowledge about infection prevention among nursing home personnel and identify gaps potentially addressable through a quality improvement collaborative. DESIGN Baseline knowledge assessment of catheter-associated urinary tract infection, asymptomatic bacteriuria, antimicrobial stewardship, and general infection prevention practices for healthcare-associated infections. SETTING Nursing homes across 14 states participating in the national "Agency for Healthcare Research and Quality Safety Program for Long-Term Care: Healthcare-Associated Infections/Catheter-Associated Urinary Tract Infection." PARTICIPANTS Licensed (RNs, LPNs, APRNs, MDs) and unlicensed (clinical nursing assistants) healthcare personnel. METHODS Each facility aimed to obtain responses from at least 10 employees (5 licensed and 5 unlicensed). We assessed the percentage of correct responses. RESULTS A total of 184 (78%) of 236 participating facilities provided 1 response or more. Of the 1,626 respondents, 822 (50.6%) were licensed; 117 facilities (63.6%) were for-profit. While 99.1% of licensed personnel recognized the definition of asymptomatic bacteriuria, only 36.1% knew that pyuria could not distinguish a urinary tract infection from asymptomatic bacteriuria. Among unlicensed personnel, 99.6% knew to notify a nurse if a resident developed fever or confusion, but only 27.7% knew that cloudy, smelly urine should not routinely be cultured. Although 100% of respondents reported receiving training in hand hygiene, less than 30% knew how long to rub hands (28.5% licensed, 25.2% unlicensed) or the most effective agent to use (11.7% licensed, 10.6% unlicensed). CONCLUSIONS This national assessment demonstrates an important need to enhance infection prevention knowledge among healthcare personnel working in nursing homes to improve resident safety and quality of care. Infect. Control Hosp. Epidemiol. 2016;1-6.