The isolated tributary hypothesis (ITH) states that headwater tributaries are insular habitats, and therefore, populations in tributaries will have low genetic connectivity. We compared genetic differentiation in a species that should have high genetic connectivity (the caddisfly, Neothremma alicia) with a species previously shown to have poorly connected populations (the flatworm, Polycelis coronata). These species should represent the range of dispersal abilities for stream invertebrates in our system, so similarly high levels of genetic differentiation among tributary populations in both species would support the ITH. We collected N. alicia from 34 stream sites, nested in 15 tributaries, and P. coronata from 50 sites, nested in 26 tributaries. Both species co-occurred in 76% of the sites from the same 4 catchments in the central Wasatch Mountains of Utah, USA. The straight-line distance between sites for N. alicia ranged from 0.5 km to 52 km, with an average of 22 km, whereas the straight-line distance between sites for P. coronata ranged from 0.5 km to 66 km, with an average of 25 km. We identified 47 haplotypes from 486 individuals of N. alicia with a 796-bp unit of mtDNA sequence data (the mitochondrial cytochrome oxidase I gene [COI]) and 130 haplotypes from 468 individuals of P. coronata with a 763-bp unit from the COI gene. All haplotypes but the dominant one of N. alicia were restricted to a single catchment, and 59.6% of N. alicia haplotypes were restricted to a single tributary. Tributaries were the only level of the stream hierarchy to show significant differentiation between N. alicia populations. In contrast, 77% of the P. coronata haplotypes occurred in a single site, and there was little gene flow either between sites in the same tributary or between tributaries. These results support the ITH because most haplotypes of both species were confined to either tributaries or sites within tributaries. Furthermore, evidence of a bottleneck event suggests that most haplotypes of N. alicia have been unable to move between tributaries for hundreds to thousands of generations. If rates of anthropogenic disturbances are high (e.g., climate warming), then the rate of population decline could exceed the rate of recolonization, increasing extinction risk for invertebrates in streams of the central Wasatch Mountains regardless of their relative dispersal abilities.