•Plague, a classified re-emerging disease requires diagnosis at early stage for timely clinical management of patients and containment of infection.•PCR-LF assay being a molecular assay can rapidly detect Y. pestis obviating the requirement of agarose gel electrophoresis for end point detection of amplicons.•The described assay detected as low as 1 pg genomic DNA of Y. pestis and also detected Y. pestis in artificially spiked blood samples.•PCR-LF is a simple assay and may prove useful for plague diagnosis in peripheral laboratories with poor resources in endemic regions of plague. Plague is a zoonotic disease caused by Yersinia pestis, a Gram-negative, rod shaped coccobacillus, which is primarily found in rodents and can be transmitted to humans through flea bite. The disease has three major clinical forms bubonic (by flea bite), pneumonic (by respiratory droplets) and septicemic plague. Y. pestis is classified as a category ‘A’ agent by NIAID, USA due to its high mortality and easy person to person dissemination. The conventional diagnostic methods available for Y. pestis show cross-reactivity with other enteropathogenic bacteria making its detection difficult. There is a need to develop sensitive and specific molecular assay for accurate detection of Y. pestis. PCR is well suited molecular biology tool for rapid diagnosis of plague but after completion of thermal cycling steps, it requires additional time to analyze amplified product using agarose gel electrophoresis. In the present study, PCR assay coupled with lateral flow strips has been developed for rapid detection of Y. pestis. Lateral flow strips give an alternative to gel electrophoresis and permit easy and rapid detection of PCR products. The PCR was performed with 5′ 6-FAM and biotin tagged primers specific for Y. pestis, targeting yihN gene located on chromosome. The PCR product was analyzed using lateral flow strips which yielded result within 2-3 minutes. The analytical sensitivity of PCR-lateral flow (PCR-LF) assay was 1 pg genomic DNA of Y. pestis and 500 copies of target DNA sequence harboured in a recombinant plasmid. The assay could detect Y. pestis DNA extracted from spiked human blood samples containing ≥104 CFU per mL of bacteria. The assay was found to be specific and did not cross react with other closely related bacterial species. The developed assay was highly specific, sensitive and also did not require agarose gel electrophoresis for post amplification analysis.