Eustrongylides excisus is a parasitic nematode species whose life cycle requires two intermediate hosts, an aquatic oligochaete and a benthophagous fish. In the present study, 100 specimens of Esox lucius Linnaeus weighing 550-800 g were collected from Freidoonkenar, south of the Caspian Sea (Mazandaran province, Iran) and were examined for the presence of Eustrongylides. The bright red nematode larvae were found in the testes and encapsulated in the body musculature of 90% of E. lucius fish. The larvae were diagnosed as Eustrongylides excisus. Tissue samples were collected from the muscles and testes for histopathological examination of the lesions caused by the parasitic larvae. For molecular analysis, the nematode larvae genomic DNA was extracted and molecular characterisation of Eustrongylides and comparison with the corresponding sequences available in the GenBank was done. The histopathological damages caused by parasites in the muscle included external nodules, inflammation, necrosis, and granulomas. Granulomas containing multi-nucleated giant cells, epithelioid cells, lymphoid cells, macrophages and necrotic debris were observed. Microscopic examination of the testes revealed mild vacuolar degeneration in some Sertoli cells. Molecular analysis confirmed obtained larvae as E. excisus. Comparison of DNA sequences showed that isolated nematodes were very similar to those obtained from freshwater fish in China. The present study reported Eustrongylides nematodes in Esox lucius, and inflammatory lesions caused by E. excisus larvae in the muscle and testis of this species of fish for the first time. In addition, molecular characterization and phylogenic analysis of recovered larvae showed presence of microvarients.