Background: Trichomonosis is one of the most common gastrointestinal infections found in pigeons worldwide. Although many studies in Iran have examined the prevalence of Trichomonas gallinae infections in pigeons and various bird species, there have been relatively few studies investigating the genotypes of this protozoan. Objectives: The purpose of this study was to examine T. gallinae infection in pigeons in the Mashhad area, Iran, using parasitological, pathological, and genetic methods. Methods: Between April and July 2022, a total of one hundred oral swabs were gathered for microscopic, culture, and molecular analysis. Additionally, deceased pigeons were transported to the laboratory for necropsy and histopathological examinations. Sequence analysis was performed on five PCR products from T. gallinae based on the amplification of the ITS1/5.8S/ITS2 gene to identify their genotype. Results: Out of 100 collected samples, T. gallinae infection was detected in 50 samples using microscopic examinations, culture, and PCR methods. Among the infected pigeons, 49 exhibited no clinical symptoms, whereas only one pigeon presented with severe clinical symptoms and ultimately succumbed to the illness. The macroscopic assessment revealed the presence of yellow caseous necrotic material in the oral cavity and pharynx. Histopathological analysis demonstrated infiltration of mononuclear cells and hyperemia within the mucosal tissues of the oral cavity and esophagus and crop, additionally, necrotic lesions and hyperemia were identified in the liver. The sequencing and phylogenetic examination of the internal transcribed spacer (ITS) region from five isolates in this study reveal a distinct lineage of T. gallinae present in pigeons located in the Mashhad area. Conclusions: The study results indicated a high prevalence of T. gallinae infection in pigeons, and for the first time, a distinct lineage of T.gallinae from pigeons in Iran was documented.

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