Technical Abstract: Several bumble bee (Bombus) species in North America have undergone range reductions and rapid declines in relative abundance. Pathogens have been suggested as causal factors, however, baseline data on pathogen distributions in a large number of bumble bee species have not been available to rigorously test this hypothesis. In a nationwide survey of the U.S., nearly 10,000 specimens of 36 bumble bee species collected at 284 sites were evaluated for the presence and prevalence of two known Bombus pathogens, the microsporidium Nosema bombi and the trypanosome Crithidia bombi. Prevalence of C. bombi was =10% for all host species examined but was recorded from 21% of surveyed sites. This pathogen was isolated from 15 of the 36 Bombus species screened, and was most commonly recovered from B. bifarius, B. bimaculatus, B. impatiens and B. mixtus. Nosema bombi was isolated from 22 of the 36 collected U.S. species; prevalence reached 37.2% in Bombus occidentalis and 15.2% in B. pensylvanicus, two species that are reportedly undergoing population declines in North America. Nosema bombi was isolated from these two hosts in more than 40% of surveyed sites, significantly more than for other hosts, and spore intensity in midgut tissues was higher than in non-declining species. Variants of a tetranucleotide repeat in the internal transcribed spacer (ITS) of the N. bombi rRNA gene, thus far not reported from European isolates, were isolated from most U.S. Bombus hosts, appearing in varying ratios in different host species with the possible exception of B. occidentalis. Given the overall genetic similarity, the presence of a unique N. bombi isolate in U.S. bumble bees does not preclude introduction of an exotic strain into North America, but it could reveal one or more native North American strains and indicate that N. bombi is enzootic across the Holarctic Region, exhibiting some genetic isolation.