Freshwater ecosystems face significant biodiversity losses, with fish species such as the Tippecanoe darter (Etheostoma tippecanoe) being particularly vulnerable due to their sensitivity to habitat degradation and pollution. Reintroduction efforts are increasingly employed to restore extirpated populations, but their success depends on the suitability of receiving habitats. This study evaluates environmental and biological parameters at reintroduction sites within the Muskingum River drainage in Ohio (USA) to determine habitat suitability for Tippecanoe darters. Six sites across four rivers (Kokosing, Walhonding, Muskingum, and Tuscarawas) were sampled for water chemistry, algae, macrophytes, macroinvertebrates, and fish communities. Multivariate analyses (CCA, NMDS, MRPP, ISA) revealed two primary site clusters. Group A (Kokosing and Walhonding Rivers) exhibited significantly higher dissolved oxygen, algal richness and density, and Tippecanoe darter abundance, alongside lower turbidity and macrophyte cover compared to Group B (Muskingum and Tuscarawas Rivers). Indicator taxa such as Hydropsychidae and Isonychiidae, known for their sensitivity and value as prey for darters, were also more prevalent in Group A. These findings suggest that smaller, cooler, and higher-quality tributaries, such as the Kokosing and Walhonding Rivers, provide more suitable conditions for Tippecanoe darter survival and reintroduction. Future analyses will include further incorporation of fish community data. This research supports the use of comprehensive bioassessment in identifying viable habitats for conservation and offers key insights for future darter reintroduction strategies.