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Abstract Enterocytozoon hepatopenaei (EHP) is an obligate intracellular microsporidium that parasitizes shrimp, causing problems such as growth retardation and immune dysfunction in its hosts. Although EHP infection has been confirmed in various penaeid shrimp, the host range of EHP across crustacean families and the mechanism underlying its potential cross-family infection remain unclear, especially regarding non-penaeid species. To explore the infection status of EHP from different crustacean families, we selected five different families, i.e., Penaeidae ( Penaeus vannamei ), Atyidae ( Neocaridina denticulata ), Palaemonidae ( Palaemon carinicauda ), Cambaridae ( Procambarus clarkii ) and Lysiosquillidae ( Oratosquilla oratoria ). Using artificial infection, in combination with molecular detection and pathological analysis, we systematically evaluated the infection characteristics of EHP in the different families. The results showed that individuals from the five families were all positive for EHP after three days of infection (dpi). qPCR, which was used to detect the change trend of EHP load (ranging from 10 1 to 10 4 copies EHPptp2/ng gDNA) in the five species, also showed a similar pattern: first increasing at 1-3 dpi, then decreasing at 5-7 dpi, and then increasing again at 10 dpi. Although the EHP loads in the different species varied, they showed consistent pathological characteristics: the longer the infection duration, the greater the degree of tissue cell damage. By 10 dpi, which corresponds to the late infection stage, proliferating spores in the hepatopancreas were detected — importantly, all species showed a significant increase in spore load at this time point. Meanwhile, the hepatopancreatic acinar structures of each family gradually disintegrated, accompanied by basement membrane rupture, epithelial cell vacuolization, and spore aggregation, ultimately leading to organ failure. Our results also confirmed that Oratosquilla oratoria and Neocaridina denticulata can host EHP, revealing for the first time the cross-family infection of EHP in crustaceans.