This study reports the results of a collaborative study undertaken by two independent research groups to (a) confirm recent PCR-based detection of Yersinia pestis DNA in human teeth from medieval plague victims in France, and (b) to extend these observations over five different European burial sites believed to contain plague victims dating from the late 13th to 17th centuries. Several different sets of primers were used, including those previously documented to yield positive results on ancient DNA extracts. No Y. pestis DNA could be amplified from DNA extracted from 108 teeth belonging to 61 individuals, despite the amplification of numerous other bacterial DNA sequences. Several methods of extracting dentine prior to the DNA extraction were also compared. PCR for bacterial 16S rDNA indicated the presence of multiple bacterial species in 23 out of 27 teeth DNA extracts where dentine was extracted using previously described methods. In comparison, positive results were obtained from only five out of 44 teeth DNA extracts for which a novel contamination-minimizing embedding technique was used. Therefore, high levels of environmental bacterial DNA are present in DNA extracts where previously described methods of tooth manipulation are used. To conclude, the absence of Y. pestis-specific DNA in an exhaustive search using specimens from multiple putative European plague burial sites does not allow us to confirm the identification of Y. pestis as the aetiological agent of the Black Death and subsequent plagues. In addition, the utility of the published tooth-based ancient DNA technique used to diagnose fatal bacteraemias in historical epidemics still awaits independent corroboration.