A spectral line-scan imaging system was developed for automated online wholesomeness inspection of broilers and evaluated in a commercial chicken processing plant. Real-time online hyperspectral images acquired by the system on a 140 bird-per-minute processing line were analyzed to optimize Region of Interest (ROI) size and location and to determine key wavebands by which to implement online high-speed multispectral inspection. Multispectral imaging algorithms were implemented to automatically recognize individual carcasses entering and exiting the field of view, to locate the ROI on the bird, and to determine the condition for each carcass as being wholesome or unwholesome. The high accuracy obtained from the in-plant evaluation results showed that the system can effectively perform food safety inspection tasks on high-speed processing lines. The system is being adapted for commercial use in pre-sorting chicken during initial processing operations, to help poultry processors improve production efficiency and satisfy increasing consumer demand for poultry products.