Invasive species are one of the major threats to biological diversity. Invasive species of crayfish are known to negatively impact native species in aquatic ecosystems. We recently found that an Ozark endemic crayfish, Orconectes neglectus, has been introduced into the Spring River drainage of southern Missouri and northern Arkansas and appears to have the potential to negatively impact the native communities. We used quantitative kick netting along the Spring River and selected tributaries to determine the distribution and abundance of O. neglectus and its potential to impact native crayfish species. The native crayfishes Orconectes eupunctus, a species of special concern, and Cambarus hubbsi appear to no longer occur throughout much of their former range in the Spring River drainage where O. neglectus is now abundant. Orconectes eupunctus, C. hubbsi, and O. neglectus mainly used fast-flowing riffle and run habitats with a mix of gravel, cobble, and boulder, whereas the other common species collected, Orconectes punctimanus and Orconectes ozarkae, were more generalists in habitat use and were found at all sampled sites. Orconectes eupunctus and C. hubbsi were positively associated with each other, but negatively associated with O. neglectus, despite their similar habitat use. These results provide evidence that O. neglectus is expanding its range, possibly to the detriment of O. eupunctus and C. hubbsi. An intensive field survey and manipulative experiments would be required to confirm the disappearance of the native species, and the mechanisms involved.