The use of molecular techniques to detect Yersinia pestis has enabled remarkable progress in the provision of necessary information on the occurrence of plague. In Tanzania, despite the long history of plague, DNA confirmation on the presence of Y. pestis in human specimens has not been done. This study was conducted in Mbulu district in Northern Tanzania where plague outbreaks have recently been reported. Nine human bubo specimens were investigated for Y. pestis plasminogen activator gene by using polymerase chain reaction (PCR), and two were found to be positive. The two positive amplicons, together with three previously obtained PCR positive rodent samples, were sequenced using a 3130 genetic analyzer and then compared with those available in GenBank by basic local alignment search tool (BLAST). All sequences obtained from both human and rodent samples showed 99% sequence similarity to Y. pestis plasmid pPCP1, detected from ancient DNA, confirming the presence of Y. pestis in humans that possibly sourced from rodents in Tanzania.