Glyphosate resistance in crop weeds is commonly attributed to rapid evolution through the amplification of the target gene, EPSPS (5-enolpyruvylshikimate-3-phosphate synthase). This amplification typically occurs through mechanisms such as unequal recombination, segmental duplications within the target chromosome, or the formation of ring chromosomes and extrachromosomal circular (ecc) DNA elements containing EPSPS. However, structural abnormalities in chromosomes not directly associated with EPSPS amplification have not been documented in the glyphosate-resistant weed population. Here, we describe the presence of a large chromosome found exclusively in the glyphosate-resistant Amaranthus tuberculatus (waterhemp) population but absent in susceptible counterparts. This large chromosome (~ 6 μm) is approximately twice the size of normal chromosomes (~ 2-3 μm) and is present in both male and female euploid plants (2n = 32) in a heteromorphic state. It aroses through pericentromeric heterochromatin expansion and duplications of the 5S rDNA locus but notably lacks the EPSPS gene. The large chromosome pairs with its normal homolog but was not transmitted to progeny in controlled greenhouse matings, suggesting a fitness cost in the absence of glyphosate selection pressure. This large chromosome offers a potential resource for the investigation of chromosome evolution of adaptive traits for glyphosate resistance in A. tuberculatus.