Abstract: Background and Objective: Weeds have exceptional capability to reproduce and evolved in response to weeding practices via modified genetic structure. Although, weeds stand for a consider concerns in the agriculture sector, it had received less attention on the molecular level. The current study designed to elucidate the evolution of geographically-isolated johnsongrass species using Random Amplified Polymorphic DNA (RAPD) and Internal Transcribed Spacer (ITS) molecular markers. Materials and Methods: Eight fresh samples of johnsongrass plants collected from different sites around Baghdad capital city. Total genomic DNA was extracted from the eight johnsongrass populations in addition to the most cultivated sorghum cultivars (Enqath and Cafair). The molecular assessment was conducted with aid of ITS and RAPD markers. Sequencing and RAPD data were statistically analyzed with MEGA6 (version 6.0) and MVSP (version 3.22) software, respectively. Results: The RAPD data revealed a polymorphism percentage of 55.8% indicating the biased genetic performance of some johnsongrass (Sorghum halepense L.) populations which demonstrating different evolution path in response to selective pressure. The ITS sequencing showed that the weed population still had a big opportunity to evolve in response to natural environment selection and/or weeding practices. Conclusion: The detected genetic relationship between domestic varieties of sorghum and wild johnsongrass populations showed that weeding practices and domestic-related species may play an important role in updating weeds gene pool.