Glyphosate-resistant Palmer amaranth is a serious problem in southern cropping systems. Much phenotypic variation is observed in Palmer amaranth populations with respect to plant growth and development and susceptibility to herbicides. This may be related to levels of genetic diversity existing in populations. Knowledge of genetic diversity in populations of Palmer amaranth may be useful in understanding distribution and development of herbicide resistance. Research was conducted to assess genetic diversity among and within eight Palmer amaranth populations collected from North Carolina and Georgia using amplified fragment length polymorphism (AFLP) markers. Pair-wise genetic similarity (GS) values were found to be relatively low, averaging 0.34. The highest and the lowest GS between populations were 0.49 and 0.24, respectively, while the highest and the lowest GS within populations were 0.56 and 0.36, respectively. Cluster and principal coordinate (PCO) analyses grouped individuals mostly by population (localized geographic region) irrespective of response to glyphosate or gender of individuals. Analysis of molecular variance (AMOVA) results when populations were nested within states revealed significant variation among and within populations within states while variation among states was not significant. Variation among and within populations within state accounted for 19 and 77% of the total variation, respectively, while variation among states accounted for only 3% of the total variation. The within population contribution towards total variation was always higher than among states and among populations within states irrespective of response to glyphosate or gender of individuals. These results are significant in terms of efficacy of similar management approaches both in terms of chemical and biological control in different areas infested with Palmer amaranth.