Background – Equine pythiosis is an emerging, devastating disease that is hard to treat. The tumour-like nodular skin masses grow rapidly and the outcome is generally fatal, and thus early diagnosis and intervention are important. Objectives – (i) To highlight the clinical, histological and haematological findings in pythiosis, and (ii) to evaluate the efficacy of direct sample multiplex-PCR targeting the single nucleotide polymorphisms within the ribosomal DNA region for detection and genotyping of Pythium insidiosum. Animals – Two hundred and twenty horses including 204 Arabian and 16 draft horses were surveyed. Methods – Case series study diagnosis was based on clinical, pathological and haematological findings typical of P. insidiosum infection, culture identification, immunohistochemical investigation and direct sample PCR. Results – The affected horses (24 of 220, 10.91%) presented with unifocal or multiple lesions on the abdomen, limbs, chest, face and mammary gland. Cases commonly had a history of access to stagnant water, ponds and intentionally flooded rice fields. Most were pregnant mares (58.33%). Histopathology revealed granulomatous reaction, blood vessel endotheliosis, heavy infiltration of eosinophils in the dermal layer, multifocal necrosis and Splendore–Hoeppli phenomenon. Unlike direct microscopy (50%) and culture (91.6%), multiplex-PCR assay iden- tified P. insidiosum (Clade II) in all tested samples. To the best of the authors knowledge, this is the first study determining a clade of P. insidiosum causing equine pythiosis in Egypt. Conclusions and clinical importance – Direct sample multiplex-PCR assay is a potential tool for the early and rapid diagnosis of equine pythiosis. It overcomes limitations associated with morphological identification and pro- vides a definitive diagnosis