We evaluated, refined, and applied well-established polymerase chain reaction (PCR) techniques for detecting Yersinia pestis DNA in fleas (mainly Oropsylla spp.) collected from prairie dog (Cynomys spp.) burrows. Based on results from PCR of avirulent Y. pestis strain A1122 DNA, we used DNA purification and primers for the plasminogen activator gene to screen field-collected fleas. We detected Y. pestis DNA in flea pools from two black-tailed prairie dog (Cynomys ludovicianus) colonies with evidence of recent plague epizootics, and from one of four white-tailed prairie dog (Cynomys leucurus) colony complexes (Wolf Creek) where evidence of epizootic plague was lacking. Relative flea abundance and occurrence of Y. pestis DNA among flea pools appeared to vary over time at Wolf Creek. Both DNA purification and primer sequences appeared to influence the likelihood of detecting Y. pestis DNA by PCR in fleas collected from prairie dog burrows in the absence of observed epizootic plague. Presence of Y. pestis plasmid DNA in fleas collected from prairie dog burrows at Wolf Creek may represent evidence that infected fleas were somehow being maintained in that system between epizootics, consistent with the hypothesized enzootic maintenance of plague in prairie dog colony complexes elsewhere.