Palmer amaranth and kochia are major problem weeds in many cropping systems in the United States. Wide acceptance of glyphosate tolerant crop technology has resulted in extensive use of glyphosate, consequently, a number of weeds including Palmer amaranth and kochia evolved resistance to glyphosate throughout the US. Within a span of 5-7 years the glyphosate resistance in these weeds has spread extensively, devastating several major crops. Understanding the mechanisms of herbicide resistance is valuable to determine the level of resistance as well as how the resistance spreads in the populations. Glyphosate resistance mechanisms in Palmer amaranth and kochia have been investigated extensively. Although resistance to glyphosate has evolved as a result of amplification of 5-enolpyruvylshikimtate-3-phosphate synthase (EPSPS), the target site of glyphosate, but the distribution and configuration of amplified copies of EPSPS gene in the genomes of these two species is different. The EPSPS gene amplification may have possibly mediated by transposons in Palmer amaranth and whereas, likely to have resulted because of unequal recombination in kochia. These findings suggest that the EPSPS amplification can occur via different mechanisms in different weeds. Evolution of glyphosate resistance as a result of EPSPS gene amplification is a threat to long-term sustainability of glyphosate-resistant crop technology.