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Bumble bees (Order Hymenoptera) are important pollinators of economically significant crops. In the past 20 years, there has been a decline in several North American species, including the federally endangered rusty patched bumble bee (Bombus affinis). Once common throughout the upper Midwest and Eastern U.S., B. affinis is now absent from 87% of its historical range. Several causal factors contributing to bumble bee decline have been identified, including habitat loss/degradation, the increased use of pesticides, and an increase in pathogens. Pathogens, particularly those originating from commercial colonies, have been implicated in this decline and are an immediate concern for conservationists. However, the combined effects of pathogen distribution and land cover on pathogen prevalence in the U.S.A is largely unknown. Madison, WI is a large metropolitan area, within the extant range of the rusty patched bumble bee and is bordered by suburbs, exurbs, agriculture, and forested areas. During the summers of 2019 and 2020, 2,094 bees were collected from 20 sites along an urban to rural gradient. Each bee was screened for the microparasites Vairimorpha spp., Crithidia bombi, Crithidia expoeki, and Apicystis bombi using a multiplex PCR panel. Conopid flies (Order Diptera) and mermithid nematodes (Order Mermithida), both large endoparasitoids, were found across sites. Bombus impatiens, the most abundant species, had the highest pathogen prevalence and the most prevalent pathogen in this species was Apicystis bombi.