Tomato is a major horticulture crop grown across the globe. Unfortunately, its yield is reduced by 25% because of auxin herbicides and glyphosate drift. In this present study, wild germplasm of tomato was screened for herbicide tolerance. From the greenhouse study nine accessions for glyphosate and 2,4-D, eleven accessions for dicamba, five accessions for quinclorac, eight accessions for aminocyclopyrachlor, and two accessions for picloram and aminopyralid were identified to be tolerant. A few accessions were selected from each herbicide tolerant group for field trials at two locations in Mississippi in 2016 and 2017. Results indicated that TOM18 was most tolerant to dicamba herbicide, while TOM87 and TOM129 to glyphosate and quinclorac herbicide, respectively, on the basis of yield and injury. Molecular experiments were conducted to measure the genetic diversity among diverse germplasm. Genetic diversity analysis showed wild accessions to be highly diverse as compared to cultivated tomato.