In Central Europe invasive North American crayfishes are carriers of the oomycete Aphanomyces astaci, which causes crayfish plague. This lethal disease currently represents one of the major threats to native European crayfishes. We used molecular methods-species-specific amplification and sequencing of the pathogen DNA-to investigate the prevalence of individuals latently infected with A. astaci in 28 populations of two invasive American crayfish species (6 of the signal crayfish [Pacifastacus leniusculus] and 22 of the spiny-cheek crayfish [Orconectes limosus]) in the Czech Republic. The pathogen occurred in 17 investigated populations. We recorded a high variation in positive reactions, ranging from 0% to 100%, in populations of O. limosus. In P. leniusculus, however, only one individual out of 124 tested positive for the pathogen. There was a clear relationship between the water body type and pathogen prevalence in O. limosus. Infection ratios in isolated standing waters were usually low, whereas in running waters, pathogen prevalence often exceeded 50%. Other evaluated characteristics of potential plague pathogen carriers (size, sex, and the presence of melanized spots in the cuticle) seemed to be unrelated to infection. Our data suggest that in contrast to other European countries, O. limosus seems to be the primary reservoir of crayfish plague in the Czech Republic. Although all populations of alien American crayfishes may be potential sources of infections and should be managed as such, knowledge on the prevalence of the plague pathogen at various localities may allow managers to focus conservation efforts on the most directly endangered populations of native crayfishes.