Crayfish have strong ecological impacts in freshwater systems, yet our knowledge of their parasites is limited. This study describes the first systemic microsporidium (infects multiple tissue types) Alternosema astaquatica n. sp. (Enterocytozoonida) isolated from a crayfish host, Faxonius virilis, using histopathology, transmission electron microscopy, gene sequencing, and phylogenetics. The parasite develops in direct contact with the host cell cytoplasm producing mature spores that are monokaryotic and ellipsoid in shape. Spores have 9–10 coils of the polar filament and measure 3.07 ± 0.26 µm (SD) in length and 0.93 ± 0.08 µm (SD) in width. Our novel isolate has high genetic similarity to Alternosema bostrichidis isolated from terrestrial beetles; however, genetic data from this parasite is restricted to a small fragment (396 bp) of the SSU gene. Additional data related to spore morphology and development, host, environment, and ecology indicate that our novel isolate is distinct from A. bostrichidis, which supports a new species description. Alternosema astaquatica n. sp. represents a novel member of the Orthosomella-like group which appears to be a set of opportunists within the Enterocytozoonida. The presence of this microsporidium in F. virilis could be relevant for freshwater ecosystems across this crayfish's broad geographic range in North America and may affect interactions between F. virilis and invasive rusty crayfish Faxonius rusticus in the Midwest USA.