Two populations of Palmer amaranth from New Mexico have been confirmed to be resistant to glyphosate. In the present study, the molecular basis of resistance and the mode of inheritance of resistance in those populations were investigated. Quantitative real-time polymerase chain reaction analysis indicated up to an eightfold increase in genomic EPSPS copy number in glyphosate resistant plants compared with susceptible plants. The relative genomic EPSPS copy number of resistant plants was positively correlated with the relative EPSPS cDNA expression levels. Eight hours after treatment with glyphosate, the shikimate accumulation levels in resistant plants were negatively correlated with the genomic EPSPS copy numbers. Multiple sequencing of the EPSPS cDNA of resistant plants did not reveal any glyphosate resistance-conferring mutations. The evaluation of F1, reciprocal F1, and F2 Palmer amaranth families indicated that resistance to glyphosate does not follow a single-gene segregation pattern. Results suggest that the EPSPS amplification is the primary molecular basis of resistance in glyphosate resistant populations of Palmer amaranth from New Mexico.