We provide evidence ofEhrlichia risticiiHolland, the agent of Potomac horse fever, in trematode stages found in aquatic insects collected from a pasture stream in northern California, using nested polymerase chain reaction (PCR) amplification and sequence analyses of the 16S rRNA, 51 kDa major antigen andgroELheat shock protein genes.E. risticiiwas detected in metacercariae found in the immatures and adults of the following insects: caddisflies (Trichoptera), mayflies (Ephemeroptera), damselflies (Odonata, Zygoptera), dragonflies (Odonata, Anisoptera), and stoneflies (Plecoptera). The prevalence ofE. risticiiwas 31.9% (n= 454 individuals) in aquatic insects (13 of 17 species were positive). Prevalence within orders was as follows: 43.5% (n= 207) in caddisflies, 15.2% (n= 92) in mayflies, 13.9% (n= 115) in damselflies, 10.0% (n= 10) in dragonflies, and 80.0% (n= 30) in stoneflies. This study demonstrates a broad intermediate host range for trematodes that act as vector forE. risticii.Insects are likely to play an important role in the epidemiology of this disease.