Establishment of Myxobolus cerebralis (Mc) resulted in declines of wild Rainbow Trout Oncorhynchus mykiss populations in streams across Colorado during the 1990s. However, the risk for establishment and spread of this parasite into high-elevation habitats occupied by native Cutthroat Trout O. clarkii was unknown. Beginning in 2003, tubificid worms were collected from all major drainages where Cutthroat Trout were endemic and were assayed by quantitative PCR to determine the occurrence and distribution of the various lineages of Tubifex tubifex (Tt) oligochaetes. Over a 5-year period, 40 groups of Tt oligochaetes collected from 27 streams, 3 natural lakes, 2 private ponds, and a reservoir were evaluated for their relative susceptibility to Mc. Exposure groups were drawn from populations of pure lineage III Tt, mixed-lineage populations where one or more of the highly resistant (lineage I) or nonsusceptible lineages (V or VI) were the dominant oligochaete and susceptible lineage III worms were the subdominant worm, or pure lineage VI Tt. Experimental replicates of 250 oligochaetes were exposed to 50 Mc myxospores per worm. The parasite amplification ratio (total triactinomyxons [TAMs] produced / total myxospore exposure) was very high among all pure lineage III Colorado exposure groups, averaging 363 compared with 8.24 among the mixed-lineage exposure groups. Lineage III oligochaetes from Mt. Whitney Hatchery in California, which served as the laboratory standard for comparative purposes, had an average parasite amplification ratio of 933 among 10 exposed replicates over a 5-year period. Lineage I oligochaetes were highly resistant to infection and did not produce any TAMs. Lineages V and VI Tt did not become infected and did not produce any TAMs. These results suggest that the risk of establishment of Mc is high for aquatic habitats in Colorado where Cutthroat Trout and lineage III Tt are sympatric.