The discovery of non-chromosomal circular DNA offers new directions in linking genome structure with function in plant biology. Glyphosate resistance through EPSPS gene copy amplification in Palmer amaranth was due to an autonomously replicating extra-chromosomal circular DNA mechanism (eccDNA). CIDER-Seq analysis of geographically distant glyphosate sensitive (GS) and resistant (GR) Palmer Amaranth (Amaranthus palmeri) revealed the presence of numerous small extra-chromosomal circular DNAs varying in size and with degrees of repetitive content, coding sequence, and motifs associated with autonomous replication. In GS biotypes, only a small portion of these aligned to the 399 kb eccDNA replicon, the vehicle underlying gene amplification and genetic resistance to the herbicide glyphosate. The aligned eccDNAs from GS were separated from one another by large gaps in sequence. In GR biotypes, the eccDNAs were present in both abundance and diversity to assemble into a nearly complete eccDNA replicon. Mean sizes of eccDNAs were similar in both biotypes and were around 5kb with larger eccDNAs near 25kb. Gene content for eccDNAs ranged from 0 to 3 with functions that include ribosomal proteins, transport, metabolism, and general stress response genetic elements. Repeat content among smaller eccDNAs indicate a potential for recombination into larger structures. Genomic hotspots were also identified in the Palmer amaranth genome with a disposition for gene focal amplifications as eccDNA. The presence of eccDNA may serve as a reservoir of genetic heterogeneity in this species and may be functionally important for survival.