Leptospirosis is a major zoonotic disease endemic in the Philippines with an emerging pattern of post-typhoon increase of epidemiological cases. In 2009 after the typhoons Ondoy and Pepeng, a large number of cases were reported in Marikina City, Metro Manila, one of the major flood sites. The incidence pattern was in contrast with the absence of cases in prior years, suggesting the emergence of a vulnerable site with climatic change. The lack of information on infectious agent reservoirs like feral rats thriving in the site presents an opportunity for testing a biosurveillance protocol where gene-based detection methods could be explored. Thus, this study was aimed to test the utility of binary gene markers, lipL32 and gyrB in the detection and identification of leptospires in feral rats in Marikina City. Traps were installed in two selected sites: Barangay Malanday and the Marikina Public Market where twenty-seven rats were recovered. The rats were dissected on site for their kidneys; genomic DNAs were extracted and assayed for leptospires by PCR amplification of lipL32 and gyrB. Results showed one rat sample from Barangay Malanday to be positive for pathogenic Leptospira spp. and subsequent sequence analysis of the amplicons indicated that the DNA sequence belongs to Leptospira interrogans. The partial gyrB sequence obtained differed from the L. interrogans strain sequences deposited in the GenBank, suggesting a possible new strain. This study reports the utility of binary (lipL32 and gyrB) gene marker analysis in the detection of pathogenic Leptospira in a sample biosurveillance of emerging infection of feral rats in Marikina City.