Trichomonas gallinae is a protozoan parasite that causes canker in pigeons. Squabs (young pigeons) are frequently infected with T. gallinae and can die because of the infection, while adult pigeons can act as carriers showing no clinical signs. In the present study, 50 squabs, up to 1-month-old, were purchased from pigeon markets in different regions of the Giza governorate, Egypt. Direct wet mount preparations of the oral excretions of the squabs (mouth wash) and Giemsa staining revealed that 64% (32/50) were positive for T. gallinae. Experimental infection of ten squabs with 103 T. gallinae trophozoites/ml resulted in oral lesions on the mouth, tongue, and soft palate, with the presence of yellowish-white nodules (cheese-like) in the oral cavity on the sixth day post-infection in all squabs. A subset of five samples were cultured in modified Diamond’s media, their DNA was extracted, and a portion of the ribosomal internal transcribed spacer region (ITS1/5.8S/ITS2) was amplified by polymerase chain reaction (PCR) followed by sequencing. Phylogenetic analysis of the five isolates revealed 64–91% homology with some reference isolates circulating in Egypt and related countries.