The ileal Peyer's patches (Pp), secondary gut-associated lymphoid tissue of the mucosal immune system, may serve as an important site for monitoring inflammatory and immunologic responses of the host against enteric pathogens. Chicken Pp are often difficult to observe grossly, and a simple technique to enhance visualization of the Pp is lacking. Therefore, we designed a novel staining method that is quick, easy, and accurate to aid in gross identification and recovery of the chicken Pp from fresh tissue specimens. Lower alimentary tracts were harvested from White Leghorn hens and commercial broilers. The ileocecocolic region was excised intact, flushed with deionized water to remove ingesta, and a dilute eosin-Y solution was infused. After 1 min, the eosin-Y was gently extruded. Modified-crystal violet (mCV) was then injected into the gastrointestinal segment, where on the lymphoid tissue area became apparent at the serosal surface. The distal ileal Pp was visible as a pale whitish pink ovoid-focalized area with surrounding gut tissue stained light purple. The exact Pp site could be delineated at the serosal and mucosal surface by gross assessment. Light microscopy evaluation of hematoxylin and eosin-stained tissue slides prepared from the excised Pp site revealed lymphoid tissue aggregations with multiple follicular units indicative of Pp. The novel eosin-Y mCV staining technique promotes rapid identification and accurate recovery of chicken Pp lymphoid tissue from fresh tissue specimens.