Orobanche are the devastating holoparasitic weeds causing extensive damage to the mustard cultivation in India. Considering the tedious species identification from a single seed and seed longevity in the soil for years, the mitigation of this weed is difficult. Therefore, development of molecular diagnostic assay specific to Orobanche aegyptiaca is required for weed management. In this study, a polymerase chain reaction (PCR) based strategy was optimised using internal transcribed spacer (ITS) based markers to identify the Orobanche species predominant in the mustard fields. Genomic DNA was extracted from tissue and soil samples artificially inoculated with seeds of Orobanche spp. and subjected to PCR analysis. ITS primers amplified a 350bp PCR product specific to O. aegyptiaca confirming its dominance in mustard crop fields of India. Furthermore, soil and tissue samples were collected from the seven farmer fields of Rajasthan and subjected to PCR analysis using ITS-350 primers. ITS-350 primers amplified all the soil/tissue samples confirming the specificity of the method and markers applied. It was also found that one Orobanche plant could attach itself to the host plant through many haustoria and also many Orobanche plants could attach to the one mustard plant through individual haustorium. This assay can also be applied to identify seed contaminants in commercial seed lots. A small soil sample taken from the mustard field can provide clues about the infestation likely to affect crop yield and productivity. Based on diagnosis suitable recommendations for crop management and input on fertilizer doses can be provided to the farmers on timely basis.