Naegleria fowleri the causative agent of Primary Amoebic Meningoencephalitis, is ubiquitously distributed worldwide in various warm aquatic environments and soil habitats. The present study reports on the presence of Naegleria spp. in various water bodies present in Rohtak and Jhajjar district, of state Haryana, India. A total of 107 water reservoirs were screened from summer till autumn (2012 and 2013). In order to isolate Naegleria spp. from the collected water samples, the water samples were filtered and the trapped debris after processing were transferred to non-nutrient agar plates already seeded with lawn culture of Escherichia coli. Out of total 107 water samples, 43 (40%) samples were positive by culture for free living amoeba after incubation for 14 days at 37°C. To identify the isolates, the ITS1, 5.8SrDNA and ITS2 regions were targeted for PCR assay. Out of total 43 positive samples, 37 isolates were positive for Naegleria spp. using genus specific primers and the most frequently isolated species was Naegleria australiensis. Out of 37 Naegleria spp. positive isolates, 1 isolate was positive for Naegleria fowleri. The sequence analysis revealed that the Naegleria fowleri strain belonged to Type 2.