Root lesion nematode (Pratylenchus spp.) is an economically important plant-parasitic nematode. The genus is difficult to identify taxonomically as species look very similar even under high magnification. This study aimed to isolate and characterize Pratylenchus species found at the Indian Agricultural Research Institute farm in New Delhi through both morphological and molecular analysis in order to authenticate identifications. Soil samples were collected from different crops and nematodes were extracted and isolated. Specimens were then studied morphologically and through analysis of the internal transcribed spacer region of ribosomal DNA.