Technical Abstract: An online line-scan imaging system containing an electron-multiplying charge-coupled device detector and line-scan spectrograph was used for identifying wholesome and unwholesome freshly slaughtered chicken carcasses on high-speed commercial chicken processing lines. Hyperspectral images were acquired using the line-scan imaging system for 5549 wholesome chicken carcasses and 93 unwholesome chicken carcasses on a commercial processing line, for analysis to optimize ROI size and location and to determine the key intensity waveband and ratio wavebands to be used for online inspection. Multispectral imaging algorithms were developed for real-time online identification of wholesome and unwholesome chicken carcasses. The imaging system inspected over 100,000 chickens on a commercial 140 bpm kill line during continuous operation and achieved over 99% accuracy in identifying wholesome chickens and over 96% accuracy in identifying unwholesome diseased chickens. A system of this type can perform food safety inspection tasks accurately and with less variation in performance at high speeds (e.g., at least 140 bpm), and help poultry plants to improve production efficiency and satisfy increasing consumer demand for poultry products.