Plague is a re-emerging zoonotic disease caused by the bacterium Yersinia pestis. The disease has caused periodic global devastation since the first outbreak in the 6th century. Two months after a suspected plague outbreak in Nyimba district, samples were collected from 94 livestock (goats and pigs), 25 rodents, 6 shrews and 33 fleas. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) techniques were used to investigate the presence of Y. pestis, which showed that 16.0% (4/25) of rodents, 16.7% (1/6) of shrews (Crocidura spp) and 6.0% (5/83) of goats were positive for IgG antibodies against Fraction 1 antigen of Y. pestis. Plasminogen activator (Pla) gene (DNA) of Y. pestis was detected in five pools containing 36.4% (12/33) fleas collected from pigs (n = 4), goats (n = 5) and rodents (n = 3). The detection of Pla gene in fleas and IgG antibodies against Fraction1 antigen in rodents, shrews and goats suggest that Y. pestis had been present in the study area in the recent past.