Phlebotomine sand flies are vectors of bacteria, parasites, and viruses. Lutzomyia apache, a North American sand fly, was incriminated as a vector of vesicular stomatitis viruses (VSV) due to overlapping ranges of the sand fly and recent outbreaks of VSV. We report on the discovery of 2 populations of L. apache in Wyoming from Albany and Fremont counties. We attempted to isolate VSV and phleboviruses from sand flies from Albany County and screened select flies by polymerase chain reaction for Bartonella and blood meals. We did not isolate viruses or detect DNA from vertebrate hosts or Bartonella. Flies were also tested for insect pathogens and other microbes. We detected a Rickettsia sp. in all flies that were examined and a parasitic protozoon, Ascogregarina sp., from the midgut of a larva. Eustigmaeus lirella, a stigmaeid mite, parasitized 2 field-caught females.