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Abstract Background Squamous cell carcinoma is the most common genital, ocular and gastric tumour in horses. Equus caballus papillomavirus type 2 (EcPV2) DNA has been detected in several studies in equine penile squamous cell carcinomas (SCCs) and precursor lesions providing evidence of a causal role of EcPV2 in equine genital SCCs. Recently, EcPV2 E6/E7 nucleic acids were also detected in equine gastric SCCs, but further studies are required to determine the role of EcPV2 infection in the pathogenesis of gastric SCC. EcPV2 nucleic acids have been rarely described in ocular SCCs and precursor lesions. Objectives To investigate the presence of EcPV2 nucleic acids with polymerase chain reaction (PCR) and in situ hybridisation (ISH) in penile hyperplasias, papillomas and SCCs in horses and to determine whether EcPV2 nucleic acids can be detected in SCCs affecting other locations, including the stomach, ocular tissues and larynx. Methods Twenty‐one archival formalin‐fixed paraffin embedded (FFPE) tissue samples, including 12 genital lesions comprising penile hyperplasias, papillomas and SCCs, 6 ocular SCCs, 2 gastric SCCs and 1 laryngeal SCC, were screened by PCR and ISH for EcPV2 E6/E7 DNA and mRNA. Archival FFPE tissue samples (eyelid and penile mucosa and preputium) from six horses without a diagnosis or history of neoplastic or papillomavirus‐associated disease were included as controls. Results EcPV2 nucleic acids were detected by PCR and ISH in all genital lesions (12/12) and gastric SCCs (2/2), in two ocular SCCs (2/6) and in one laryngeal SCC (1/1). In control horses, one eyelid sample was positive in PCR but not in ISH. The remaining control samples were negative for EcPV2 E6/E7 nucleic acids in PCR and ISH. Conclusions These results further support the role of EcPV2 infection in the development of equine genital SCCs and suggest that EcPV2 infection may also act as a predisposing factor for other SCCs in horses, including gastric, ocular and laryngeal SCCs.