Silverleaf nightshade (Solanum elaeagnifolium Cav.) is an invasive species that has successfully spread outside its native range to become a noxious weed in 21 states in the United States and 42 countries worldwide. The successful establishment of S. elaeagnifolium outside its native habitat indicates its innate ability to adapt to a multitude of environments. Phenotypic plasticity and/or genetic adaptation have been identified as key mechanisms underlying the adaptive success of invasive species. Whereas phenotypic plasticity allows a species to buffer changes in the environment by altering its phenotypic attributes within the short term, genetic adaptation is responsible for the longer-term adaptability of plants to heterogeneous environments and is dependent on the amount of genetic variation present in the species. In this study, we screened DNA markers that are specific to tomato (Solanum lycopersicum L.) and Solanum lycopersicoides Dunal for their interspecific transferability to S. elaeagnifolium and determined the applicability of the transferable DNA markers in assessing the extent of genetic variation in populations from Lubbock, Littlefield, and Blackwell, TX. Of the 187 markers screened, 78 successfully amplified targets in S. elaeagnifolium, indicating the evolutionary conservation of marker loci across S. lycopersicum, S. lycopersicoides, and S. elaeagnifolium, despite their genetic divergence millions of years ago. Genotyping of S. elaeagnifolium populations using 50 DNA markers that consistently amplified clear bands in more than 60% of the plants identified nine polymorphic markers with 0.014 to 0.621 polymorphism information content. Genetic diversity analysis by DNA marker profiling established genetic variation among populations and within individuals of different populations. Unweighted paired group method with arithmetic mean analysis grouped the plants into six clusters that are generally defined by selection pressures unique to each collection site. Results of the study indicate the capacity of S. elaeagnifolium for genetic differentiation in response to variable selection pressures within the same geographic region.